Isolated human lung progenitor cells and uses thereof

ABSTRACT

Provided herein are methods and compositions relating, in part, to the generation of human progenitor cells committed to the lung lineage and uses of such cells for treatment of lung diseases/disorders or injury to the lung. Whether an adult stem cell can be isolated from human adult lung remains controversial in the art and at present, methods for isolating and using adult lung stem cells from humans lack reproducibility. Thus, the methods and compositions described herein are advantageous over the present state of knowledge in the art and permit the generation of human lung progenitor cells for treatment, tissue engineering, and screening assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 14/371,627, filed on Jul. 10, 2014, which is a 35 U.S.C. § 371National Stage Application of International Application No.PCT/US2013/021186, filed on Jan. 11, 2013, which designates the UnitedStates, and which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/619,568, filed on Apr. 3, 2012, and U.S.Provisional Application No. 61/586,551, filed on Jan. 13, 2012, thecontents of each of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 13, 2017, isnamed 030258-072937-US-DIV_SL.txt and is 630 bytes bytes in size.

FIELD OF THE INVENTION

The field of the invention relates to isolated human lung progenitorcells, methods of making such isolated human lung progenitor cells anduses thereof.

BACKGROUND

Lung diseases, including respiratory diseases, are a major cause ofmortality and morbidity worldwide. Current treatments are directed toreducing symptoms of lung disease and offer little to no prospect ofcure or complete disease reversal.

Transplantation of pulmonary progenitor cells derived from stem cells isone approach that can be used to regenerate endogenous lung cellsdestroyed by injury and disease. Considerable interest has developed inthe potential use of stem cells to repair lung epithelium destroyed byinjury and disease.

Stem cells represent unique cell populations that have the ability toundergo both self-renewal and differentiation. It is beneficial to beable to isolate and purify precursor cells from a subject that can bemanipulated before being reintroduced into the subject for treatmentpurposes. The use of a subject's own cells would obviate the need toemploy adjunct immunosuppressive therapy, thereby maintaining thecompetency of the subject's immune system. For example, the directeddifferentiation of induced pluripotent stem cells generated from asubject's somatic cell sample provides advantages in providingpopulations of cells for autologous regenerative cell therapy.

SUMMARY

Cellular differentiation is a complex process typically occurringthrough many cell divisions. A partially or fully differentiated cellcan be derived from a multipotent cell which itself is derived from amultipotent cell, and so on. While each of these multipotent cells canbe considered stem cells, the range of cell types each can give rise tocan vary considerably. Some differentiated cells also have the capacityto give rise to cells of greater developmental potential (e.g.,reprogramming). Such capacity can be natural or can be inducedartificially upon treatment with various factors. In many biologicalinstances, stem cells are also “multipotent” because they can produceprogeny of more than one distinct cell type, but this is not requiredfor “stem-ness.”

In addition to the capacity to differentiate to a more specificdevelopmental phenotype, self-renewal is another classical part of thestem cell definition. In theory, self-renewal can occur by either of twomajor mechanisms. Stem cells may divide asymmetrically, with onedaughter retaining the stem state and the other daughter expressing somedistinct other specific function and phenotype. Alternatively some ofthe stem cells in a population can divide symmetrically into two stems,thus maintaining some stem cells in the population as a whole, whileother cells in the population give rise to differentiated progeny only.Formally, it is possible that cells that begin as stem cells mightproceed toward a differentiated phenotype, but then “reverse” andre-express the stem cell phenotype, a term often referred to as“dedifferentiation” or “reprogramming” or “retrodifferentiation” bypersons of ordinary skill in the art. Reversal of the differentiationphenotype in this manner generally requires artificial manipulation ofthe cell, for example, by expressing stem cell specific mRNA or proteins(e.g., c-Myc, Klf4, Oct4, Sox2, among others) or by contacting a cellwith a de-differentiation medium.

The methods and compositions provided herein relate, in part, to thegeneration of human progenitor cells committed to the lung lineage anduses of such cells for treatment of lung diseases/disorders or injury tothe lung. Whether an adult stem cell can be isolated from human adultlung remains controversial in the art and at present, methods forisolating and using adult lung stem cells from humans lackreproducibility. Thus, the methods and compositions described herein areadvantageous over the present state of knowledge in the art and permitthe generation of human lung progenitor cells for treatment, tissueengineering, and screening assays.

One aspect disclosed herein relates to an isolated human Nkx2.1positive, Sox2 positive proximal airway multipotent progenitor cell, oran enriched population of human Nkx2.1 positive, Sox2 positive proximalairway multipotent progenitor cells.

In one embodiment of this aspect, the cell is Tuj1 negative and Pax8negative.

In another embodiment of this aspect, the cell is proliferative anddifferentiates into an airway basal stem cell, a ciliated cell, a Claracell, a neuroendocrine cell, or a squamous epithelial cell under chosendifferentiation conditions.

Also provided herein in another aspect is an isolated human Nkx2.1positive, Sox9 positive, distal multipotent lung progenitor cell.

In one embodiment of this aspect, the cell is FoxP2 positive and/or ID2positive. In another embodiment, the cell is ETV4/5 positive.

In another embodiment of this aspect, the cell is proliferative anddifferentiates into any epithelial lung cell when placed under chosendifferentiation conditions.

In another embodiment of this aspect, the cell differentiates into anairway basal stem cell, a ciliated cell, a Clara cell, a mucin secretinggoblet cell, a type I pneumocyte, a type II pneumocyte, a squamousepithelial cell, a bronchioalveolar stem cell, a bronchioalveolar ductjunction stem cell, a migratory CK14+ cell, or a neuroendocrine cellwhen placed under chosen differentiation conditions.

Another aspect described herein relates to an isolated human Nkx2.1positive, p63 positive multipotent airway basal stem cell, or anenriched population of isolated human Nkx2.1 positive, p63 positivemultipotent airway basal stem cells.

In another embodiment of this aspect, the cell is proliferative anddifferentiates into a ciliated cell, a Clara cell, a mucin secretinggoblet cell, or a basal cell when placed under chosen differentiationconditions.

In another embodiment of this aspect, the multipotent airway basal stemcell does not express a Clara cell marker, a ciliated cell marker, aneuroendocrine cell marker, or a squamous cell marker.

In another embodiment of this aspect, the cell is Sox2 positive.

In another embodiment of this aspect, the cell is CK5 positive and/orNGFR positive.

In another embodiment of this aspect and all other aspects describedherein, the cell is a disease-specific cell.

Another aspect provided herein relates to a composition comprising anisolated human Nkx2.1 positive, Sox2 positive proximal airwaymultipotent progenitor cell and a scaffold.

In another embodiment of this aspect, the scaffold is implantable in asubject.

In another embodiment of this aspect, the cell is autologous to thesubject into which the composition is being implanted.

In another embodiment of this aspect, the scaffold is biodegradable.

In another embodiment of this aspect, the scaffold comprises a naturalfiber, a synthetic fiber, decellularized lung tissue, or a combinationthereof.

In another embodiment of this aspect, the natural fiber is selected fromthe group consisting of collagen, fibrin, silk, thrombin, chitosan,chitin, alginic acid, hyaluronic acid, and gelatin.

In another embodiment of this aspect, the synthetic fiber is selectedfrom the group consisting of: representative bio-degradable aliphaticpolyesters such as polylactic acid (PLA), polyglycolic acid (PGA),poly(D,L-lactide-co-glycolide) (PLGA), poly(caprolactone), diol/diacidaliphatic polyester, polyester-amide/polyester-urethane,poly(valerolactone), poly(hydroxyl butyrate), polybutylene terephthalate(PBT), polyhydroxyhexanoate (PHH), polybutylene succinate (PBS), andpoly(hydroxyl valerate).

In another embodiment of this aspect, the proximal airway multipotentprogenitor cell is Tuj1 negative and/or Pax8 negative.

Another aspect described herein relates to a composition comprising anisolated human Nkx2.1 positive, Sox9 positive, distal multipotent lungprogenitor cell and a scaffold.

In one embodiment of this aspect, the multipotent lung progenitor cellis FoxP2 positive and/or ID2 positive. In another embodiment, the cellis ETV4/5 positive.

In another embodiment of this aspect, the scaffold is implantable in asubject.

In another embodiment of this aspect, the cell is autologous to thesubject into which the composition is being implanted.

In another embodiment of this aspect, the scaffold is biodegradable.

In another embodiment of this aspect, the scaffold comprises a naturalfiber, a synthetic fiber, decellularized lung, or a combination thereof.

In another embodiment of this aspect, the natural fiber is selected fromthe group consisting of collagen, fibrin, silk, thrombin, chitosan,chitin, alginic acid, hyaluronic acid, and gelatin.

In another embodiment of this aspect, the synthetic fiber is selectedfrom the group consisting of: representative bio-degradable aliphaticpolyesters such as polylactic acid (PLA), polyglycolic acid (PGA),poly(D,L-lactide-co-glycolide) (PLGA), poly(caprolactone), diol/diacidaliphatic polyester, polyester-amide/polyester-urethane,poly(valerolactone), poly(hydroxyl butyrate), polybutylene terephthalate(PBT), polyhydroxyhexanoate (PHH), polybutylene succinate (PBS), andpoly(hydroxyl valerate).

Another aspect described herein relates to a composition comprising anisolated human Nkx2.1 positive, p63 positive multipotent airway basalstem cell and a scaffold.

In one embodiment of this aspect, the airway basal stem cell is CK5positive and/or NGFR positive.

In another embodiment of this aspect, the scaffold is implantable in asubject.

In another embodiment of this aspect, the cell is autologous to thesubject into which the composition is being implanted.

In another embodiment of this aspect, the scaffold is biodegradable.

In another embodiment of this aspect, the scaffold comprises a naturalfiber, a synthetic fiber, decellularized lung tissue, or a combinationthereof.

In another embodiment of this aspect, the natural fiber is selected fromthe group consisting of collagen, fibrin, silk, thrombin, chitosan,chitin, alginic acid, hyaluronic acid, and gelatin.

In another embodiment of this aspect, the synthetic fiber is selectedfrom the group consisting of: representative bio-degradable aliphaticpolyesters such as polylactic acid (PLA), polyglycolic acid (PGA),poly(D,L-lactide-co-glycolide) (PLGA), poly(caprolactone), diol/diacidaliphatic polyester, polyester-amide/polyester-urethane,poly(valerolactone), poly(hydroxyl butyrate), polybutylene terephthalate(PBT), polyhydroxyhexanoate (PHH), polybutylene succinate (PBS), andpoly(hydroxyl valerate).

In another embodiment of this aspect and all other aspects describedherein, the cell is a disease-specific cell.

Also provided herein in another aspect are methods of generating a humanlung progenitor cell or population of human lung progenitor cells thatis Nkx2.1 positive, Tuj1 negative and Pax8 negative, the methodcomprising contacting a human foregut endoderm cell with FGF2, WNT andBMP4, each for a time and at a concentration sufficient to permitdifferentiation of said human foregut endoderm cell to an Nkx2.1positive, Tuj1 negative, Pax8 negative lung progenitor cell.

In one embodiment of this aspect, the contacting step is performed forat least 2 days.

In another embodiment of this aspect, the method further comprisescontacting the Nkx2.1 positive, Tuj1 negative, Pax8 negative lungprogenitor cell with BMP7, FGF7, a WNT antagonist, and a MAPKK/ERKantagonist, each for a time and at a concentration sufficient to permitdifferentiation of the Nkx2.1 positive, Tuj1 negative, Pax8 negativelung progenitor cell to a Nkx2.1 positive, Sox2 positive proximal airwaymultipotent progenitor cell.

In another embodiment of this aspect, the Wnt antagonist comprisesIWR-1.

In another embodiment of this aspect, the MAPKK/ERK antagonist comprisesPD98059.

In another embodiment of this aspect, the contacting step is performedfor at least 4 days.

In another embodiment of this aspect, the method further comprisescontacting the Nkx2.1 positive, Tuj1 negative, Pax8 negative lungprogenitor cell with BMP7, FGF7, a WNT antagonist, and a MAPKK/ERKantagonist each for a time and at a concentration sufficient to permitdifferentiation of the Nkx2.1 positive, Tuj1 negative, Pax8 negativelung progenitor cell to an Nkx2.1 positive, Sox9 positive distalmultipotent lung progenitor cell.

In another embodiment of this aspect, the Wnt antagonist comprisesIWR-1.

In another embodiment of this aspect, the MAPKK/ERK antagonist comprisesPD98059.

In another embodiment of this aspect, the contacting step is performedfor at least 4 days.

In another embodiment of this aspect, the culture of foregut endodermcells are derived from embryonic stem (ES) cells or induced pluripotentstem cells (iPSCs).

In another embodiment of this aspect, the method further comprisescontacting a culture of Nkx2.1 positive, Tuj1 negative, Pax8 negativecells with B27, BMP7, FGF7, and a WNT antagonist, each for a time and ata concentration sufficient to permit differentiation of said Nkx2.1positive, Tuj1 negative, Pax8 negative lung progenitor cell to an Nkx2.1positive, p63 positive multipotent airway basal stem cell.

In another embodiment of this aspect, the method further comprisescontacting the culture of Nkx2.1 positive, Tuj1 negative, Pax8 negativecells with Noggin.

In another embodiment of this aspect, the contacting step is performedfor at least 10 days.

Also provided herein in another aspect, are methods for treating a lungdisease or disorder, or lung injury in a subject, the method comprising:administering a composition comprising an isolated human lung progenitorcell and a pharmaceutically acceptable carrier to a subject having alung disease or disorder, or lung injury.

In one embodiment of this aspect, the isolated human lung progenitorcell is selected from the group consisting of: an Nkx2.1 positive, Sox2positive proximal airway multipotent progenitor cell; an Nkx2.1positive, Sox9 positive distal multipotent lung progenitor cell, anNkx2.1 positive, p63 positive multipotent airway basal stem cell, or adifferentiated cell thereof.

In another embodiment of this aspect, the proximal airway multipotentprogenitor cell is Tuj1 negative and/or Pax8 negative.

In another embodiment of this aspect, the distal multipotent lungprogenitor cell is FoxP2 and/or ID2 positive. In another embodiment, thecell is ETV4/5 positive.

In another embodiment of this aspect, the airway basal stem cell is CK5positive and/or NGFR positive.

In another embodiment of this aspect, the composition is administered tothe lung.

In another embodiment of this aspect, the isolated human lung progenitorcell is autologous to the subject to which the composition is beingadministered.

In another embodiment of this aspect, the composition further comprisesa scaffold.

In another embodiment of this aspect, the scaffold is implantable in asubject.

In another embodiment of this aspect, the scaffold is biodegradable.

In another embodiment of this aspect, the scaffold comprises a naturalfiber, a synthetic fiber, decellularized lung tissue, or a combinationthereof.

In another embodiment of this aspect, the scaffold comprises an agentthat promotes differentiation of the isolated human lung progenitorcell.

In another embodiment of this aspect, the composition is formulated foraerosol delivery.

Another aspect provided herein relates to methods of screening for anagent to treat a lung disease or disorder, the method comprising: (a)culturing a population of human disease-specific airway cells producedby in vitro differentiation of a human disease specific lung progenitorcell in the presence and absence of a candidate agent for treating alung disease or disorder, (b) comparing the expression or activity of atleast one marker that is upregulated in the disease or comparing theexpression or activity of at least one marker that is downregulated inthe disease in the presence and absence of the candidate agent, whereina decrease in the expression or activity of at least one upregulateddisease marker or an increase in the expression or activity of at leastone downregulated disease marker identifies the candidate agent as acandidate for the treatment of the lung disease or disorder in asubject.

In one embodiment of this aspect, the method further comprises stepsbefore step (a) of differentiating a population of isolated humandisease-specific lung progenitor cells to a culture of humandisease-specific airway cells.

In another embodiment of this aspect, the method further comprises stepsbefore step (a) of differentiating a population of induced pluripotentstem cells derived from a subject having a lung disease or disorder to apopulation of isolated human disease-specific lung progenitor cells.

In another embodiment of this aspect, the candidate agent comprises asmall molecule, a protein, a polypeptide, an antibody or an antigenbinding fragment thereof, or a nucleic acid.

In another embodiment of this aspect, the human lung progenitor cell isselected from the group consisting of: a human Nkx2.1 positive, Sox2positive proximal airway multipotent progenitor cell, a human Nkx2.1positive, Sox9 positive distal multipotent lung progenitor cell, and ahuman Nkx2.1 positive, p63 positive multipotent airway basal stem cell.

In another embodiment of this aspect, the human Nkx2.1 positive, Sox2positive proximal airway multipotent progenitor cell, and the humanNkx2.1 positive, Sox9 positive distal multipotent lung progenitor cellare made by a method comprising contacting an Nkx2.1 positive, Tuj1negative, Pax8 negative lung progenitor cell with BMP7, FGF7, a WNTantagonist, and a MAPKK/ERK antagonist each for a time and at aconcentration sufficient to permit the Nkx2.1 positive, Tuj1 negative,Pax8 negative lung progenitor cell to differentiate to an Nkx2.1positive, Sox9 positive proximal airway multipotent progenitor cell, orto an Nkx2.1 positive, Sox9 positive distal multipotent lung progenitorcell.

In another embodiment of this aspect, the contacting step is performedfor at least 4 days.

In another embodiment of this aspect, the human Nkx2.1 positive, p63positive multipotent airway basal stem cell is made by a methodcomprising contacting an Nkx2.1 positive, Tuj1 negative, Pax8 negativecell with B27, BMP7, FGF7, and a WNT antagonist, each for a time and ata concentration sufficient to permit differentiation of said Nkx2.1positive, Tuj1 negative, Pax8 negative lung progenitor cell to an Nkx2.1positive, p63 positive multipotent airway basal stem cell.

In another embodiment of this aspect, the method further comprisescontacting the Nkx2.1 positive, Tuj1 negative, Pax8 negative cell withNoggin.

In another embodiment of this aspect, the contacting step is performedfor at least 10 days.

Another aspect provided herein relates to methods of screening for anagent to induce differentiation of a human lung progenitor cell, themethod comprising: (a) culturing an Nkx2.1 positive, Tuj1 negative, Pax8negative human lung progenitor cell in the presence and absence of acandidate differentiation agent, (b) comparing the expression oractivity of at least one marker that is upregulated duringdifferentiation of the lung progenitor cell to a more differentiatedstate or comparing the expression or activity of at least one markerthat is downregulated during differentiation of the lung progenitor cellto a more differentiated state in the presence and absence of thecandidate agent, wherein a decrease in the expression or activity of atleast one upregulated differentiation marker or an increase in theexpression or activity of at least one downregulated differentiationmarker is indicative that the candidate agent can be used to inducedifferentiation of an isolated human lung progenitor cell in a subject.

In one embodiment of this aspect, the method further comprises stepsbefore step (a) of differentiating an embryonic stem cell or inducedpluripotent stem cell to a human lung progenitor cell.

In another embodiment of this aspect, the candidate agent comprises asmall molecule, a protein, a polypeptide, an antibody, or a nucleicacid.

In another embodiment of this aspect, the human lung progenitor cell isselected from the group consisting of: a human Nkx2.1 positive, Sox2positive proximal airway multipotent progenitor cell, a human Nkx2.1positive, Sox9 positive distal multipotent lung progenitor cell, and ahuman Nkx2.1 positive, p63 positive multipotent airway basal stem cell.

In another embodiment of this aspect, the human Nkx2.1 positive, Sox2positive proximal airway multipotent progenitor cell, and the humanNkx2.1 positive, Sox9 positive distal multipotent lung progenitor cellare made by a method comprising contacting an Nkx2.1 positive, Tuj1negative, Pax8 negative human lung progenitor cell with BMP7, FGF7, aWNT antagonist, and a MAPKK/ERK antagonist each for a time and at aconcentration sufficient to permit the Nkx2.1 positive, Tuj1 negative,Pax8 negative lung progenitor cell to differentiate to an Nkx2.1positive, Sox9 positive proximal airway multipotent progenitor cell, orto an Nkx2.1 positive, Sox9 positive distal multipotent lung progenitorcell.

In another embodiment of this aspect, the contacting step is performedfor at least 4 days.

In another embodiment of this aspect, the human Nkx2.1 positive, p63positive multipotent airway basal stem cell is made by a methodcomprising contacting an Nkx2.1 positive, Tuj1 negative, Pax8 negativecell with B27, BMP7, FGF7, and a Wnt antagonist, each for a time and ata concentration sufficient to permit differentiation of the Nkx2.1positive, Tuj1 negative, Pax8 negative lung progenitor cell to an Nkx2.1positive, p63 positive multipotent airway basal stem cell.

In another embodiment of this aspect, further comprising contacting theNkx2.1 positive, Tuj1 negative, Pax8 negative cell with Noggin.

In another embodiment of this aspect, the contacting step is performedfor at least 10 days.

Another aspect provided herein relates to kits for treating a lungdisease or disorder, the kit comprising: a cell of claim 1, 5, or 9, apharmaceutically acceptable carrier, and instructions for treating alung disease or disorder.

In one embodiment of this aspect, the kit further comprises a scaffold.

Also provided herein, in another aspect, is a kit for screening acandidate agent, the kit comprising: a cell of claim 1, 5, or 8, one ormore agents for detecting lung specific cell surface markers, andinstructions therefore.

In one embodiment of this aspect, the kit further comprises a cellculture medium, a growth factor, and/or a differentiation agent.

Another aspect provided herein relates to a kit(s) for differentiating ahuman stem cell to a human lung progenitor cell, the kit comprising: (i)two or more of BMP4, FGF2, WNT, BMP7, FGF7, a WNT antagonist, Noggin,B27, and retinoic acid, optionally provided in unit doses; (ii)optionally, a cell culture medium; (iii) one or more agents fordetecting a lung cell-specific surface marker; and (iii) instructionstherefore.

Also provided herein is a cell or tissue culture medium for generatingdefinitive endoderm from an iPSC or ESC, comprising: B27, Activin A, andZSTK474. In one embodiment, the medium comprises B27 in a concentrationof 1%-5% (e.g., 2%). In another embodiment, the medium comprises ActivinA in a concentration of 10-40 ng/mL (e.g., 20 ng/mL). In anotherembodiment, the medium comprises ZSTK474 in a concentration of 0.2-0.5μM. In another embodiment, the base of the medium comprises thecomponents of RPMI medium.

Also provided herein is a cell or tissue culture medium for generatingan Nkx2.1 positive lung progenitor cell, comprising: CHIR9902, PIK-75,Dorsomorphin, and FGF2. In one embodiment, the medium comprises CHIR9902in a concentration of 0.1-1 μM. In another embodiment, the mediumcomprises PIK-75 in a concentration of 0.01-0.1 μM. In anotherembodiment, the medium comprises dorsomorphin in concentration of 1-5μM. In another embodiment, the medium comprises, FGF2 in a concentrationof 10-100 ng/mL. In another embodiment, the medium further comprises adrug selected from the group consisting of: GF-109203X, Ro31-8220,Pp242, PIK-75, ZSTK474, PMA, carvedilol, corticosterone,triclabendazole, benproperine phosphate, phenothiazine, andmethotrexate.

Also provided herein is a method of generating a human lung progenitorcell or population of human lung progenitor cells that is Nkx2.1positive, Tuj1 negative, and Pax8 negative, the method comprisingcontacting a human foregut endoderm cell with a Wnt agonist, a PIK3kinase inhibitor, a BMP antagonist, and a drug selected from the groupconsisting of GF-109203X, Ro31-8220, Pp242, PIK-75, ZSTK474, PMA,carvedilol, corticosterone, triclabendazole, benproperine phosphate,phenothiazine, and methotrexate, each for a time and at a concentrationsufficient to permit differentiation of said human foregut endoderm cellto an Nkx2.1 positive, Tuj1 negative, Pax8 negative lung progenitorcell.

In one embodiment, the contacting step is performed for at least 2 days.

In another embodiment, the Wnt agonist comprises CHIR9902. In anotherembodiment, the concentration of CHIR9902 is in the range of 0.1-1 μM.

In another embodiment, the PI3 kinase inhibitor comprises PIK-75. Inanother embodiment, the concentration of PIK-75 comprises 0.01-0.1 μM.

In another embodiment, the BMP antagonist comprises Dorsomorphin. Inanother embodiment, the concentration of dorsomorphin comprises 1-5 μM.

In another embodiment, the growth factor comprises FGF2. In anotherembodiment, the concentration of FGF2 comprises 10-100 ng/mL.

Also provided herein in another aspect is a method for generating adefinitive endoderm cell or population of definitive endoderm cells, themethod comprising contacting an iPSC or ESC with B27, Activin A, andZSTK474, each for a time and at a concentration sufficient to permitdifferentiation of the iPSC or ESC to a definitive endoderm cell.

In one embodiment, generation of a definitive endoderm cell isdetermined by FOXA2/SOX17 co-staining or by FACS analysis withcKit/CXCR4 and/or cKit/EpCAM combination.

In another embodiment, the concentration of B27 comprises 1%-5% (e.g.,2%).

In another embodiment, the concentration of Activin A is 10-40 ng/mL(e.g., 20 ng·mL

In another embodiment, the concentration of ZSTK474 is 0.2-0.5 μM.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depicting an overall strategy for the step-wisedifferentiation of airway progenitors from mouse ESCs. The schematicshows the approach to develop an efficient and reproducible protocol toproduce embryonic airway epithelial progenitors from mouse ESCs. Thesignaling network promotes the differentiation of ESCs into definitiveendoderm, the anteriorization of endoderm into foregut endoderm, theinduction of the earliest lung endoderm from foregut endoderm, and thegeneration of embryonic airway progenitor cells.

FIG. 2A and FIG. 2B show data indicating that anteriorization ofendoderm to foregut endoderm promotes Nkx2.1+ cell differentiation. FIG.2A shows administration of 20 ng/ml BMP4, 20 ng/ml FGF2 and 5 nM GSK3iXVto definitive endoderm resulted in hindgut differentiation with CDX2expression, and minimal NKX2.1 induction. Scale bar, 50 μm. FIG. 2Bshows the treatment of definitive endoderm with different combinationsof BMP4/TGFβ agonists and antagonists as listed in the table for 2 days.The Foxa2+/Sox2+ cells were quantified as a percentage of positive cellsout of the total number of Foxa2+ cells. The cells were further treatedwith 20 ng/ml BMP4, 20 ng/ml FGF2 and 5 nM GSK3iXV to induce NKX2.1expression. The percentage of Nkx2.1+ cells was quantified as apercentage of the total cells present. All data were averaged from 3independent experiments.

FIG. 3A, FIG. 3B, and FIG. 3C show that BMP4, FGF2 and WNT signaling arenecessary for lung specification from foregut endoderm cells. FIG. 3A isa schematic depicting the strategy and time frame to generate Nkx2.1+cells from foregut endoderm cells. FIG. 3B is a list of variouscombinations of BMP4, FGF2, WNT and their antagonists for Nkx2.1+induction. FIG. 3C shows the NKX2.1 percentage as scored by the numberof Nkx2.1+ cells out of the total cell number in an average of 3independent experiments.

FIG. 4 shows that Smad-dependent BMP signaling is required for lungdifferentiation. NKX2.1 expression was induced with 20 ng/ml FGF2 and 5nM GSK3iXV only (control) or with additional BMP4 (20 ng/ml), BMP2 (20ng/ml), BMP7 (20 ng/ml), BMP4 (20 ng/ml)+10 μM Dorsomorphin, or BMP4 (20ng/ml)+1 μM PD98059 (data not shown). FIG. 4 shows that signaling ofBMPs through SMAD-dependent pathways is required for lung Nkx2.1+differentiation. In contrast, signaling through the MAP kinase pathwayis not required for Nkx2.1 expression. Dorsomorphin inhibits BMPsignaling through the SMAD pathway while PD98059 inhibits MAPKsignaling.

FIG. 5A and FIG. 5B show the generation of embryonic airway progenitorsfrom multipotent lung endoderm cells. FIG. 5A is a schematic depicting astrategy and time line to generate embryonic airway progenitors frommultipotent lung endoderm cells. FIG. 5B is a summary of signalingswitches that are distinct in the proximal airway and the distal lungbud tip during the pseudoglandular stage of lung development.Immunofluorescence staining of NKX2.1 and SOX2 was performed aftertreatment of Nkx2.1+ lung endoderm cells at D9 with medium containingRA-supplemented B27, 20 ng/ml BMP7, 20 ng/ml FGF7, 100 nM IWR-1 (WNTantagonist), and 100 ng/ml Noggin for 2 days (data not shown). Inaddition, immunofluorescence staining of NKX2.1 and SOX2 was performedafter treatment of Nkx2.1+ lung endoderm cells at D9 with mediumcontaining RA-supplemented B27, 20 ng/ml BMP7, 20 ng/ml FGF7, 100 nMIWR-1 (WNT antagonist), and 1 μM PD98059 for 2 day. Scale bar, 50 μm(data not shown). Data from the immunofluorescence staining indicatedthe presence of a subpopulation of Nkx2.1+ cells positive for P63 (datanot shown).

FIG. 6 is a schematic depicting an exemplary strategy and time line togenerate Nkx2.1+ lung multipotent progenitors from human iPSCs. Datafrom the experiment indicate a step-wise differentiation of Nkx2.1+ lungprogenitors from human iPSCs. A high yield of definitive endoderm fromCF1 RiPS cells was obtained after treatment for 4 days in RPMI-1640media in the presence of 2% B27 supplement, Activin A (100 ng/ml) and 5μM PI3 Kinase inhibitor LY294002 with more than 90% of cellsco-expressing transcription factors SOX17 and FOXA2 (data not shown).Anteriorization of endoderm into foregut endoderm cells was observed bydetecting SOX2 expression in Foxa2+ cells derived from CF1 RiPS cellsafter 4 days of treatment with 500 nM A-83-01 (TGFβ antagonist) and 100ng/ml Noggin (BMP4 antagonist) (data not shown). NKX2.1 staining wasperformed after anteriorization to foregut cells at D8 with serum-freemedium containing 20 ng/ml BMP4, 20 ng/ml FGF2 and 5 nM GSK3iXV for 4days (data not shown). Immunofluorescence showed co-staining ofNkx2.1-positive cells with SOX2, SOX9, TUJ1 and PAX8, therebydemonstrating a lack of thyroid and neuronal differentiation and thepresence of multipotent distal tip progenitors and Nkx2.1+/Sox2+ airwayprogenitors (data not shown). In addition, confocal images followingimmunofluorescence staining indicates some Nkx2.1+ sphere contain basalcells positive for p63 (data not shown).

FIG. 7 is a schematic depicting a treatment strategy and time frame forgenerating definitive endoderm. Immunofluorescence of FOXA2 and SOX17was performed at day 5 and indicates that >90% of cells were definitiveendoderm generated from p2A mESCs and from V6.5 mESCs (data not shown).

FIG. 8 is a table and a schematic that summarize immunofluorescence datafor specific combinations of markers segregating Nkx2.1+ brain, thyroidand lung organ anlage in mouse embryos. Immunofluorescence imaging ofNkx2.1 and SOX2 was performed in ventral forebrain, thyroid, and lung atE8.75-E9 (data not shown). Immunofluorescence imaging was also performedfor Nkx2.1, Tuj1, and Sox9 in ventral forebrain, as well as Nkx2.1, Pax8and Sox9 in thyroid at E8.75-E9.

FIG. 9A and FIG. 9B are graphs depicting a gene expression analysis ofESC-derived anterior endoderm fates. Quantitative expression of Nkx2.1,Sox2, FoxN1, Pax9, Tbx1, Pax8, Sox9 and FoxP2 mRNA in mouse ESC or humaniPSCs, corresponding definitive endoderm (DE) and anterior endoderm (AE)and AE after treatment with Nkx2.1-inductive growth factor cocktail. n=3biological triplicate replicates, the expression level is normalized tothe level of ESCs or iPSCs.

FIG. 10 is a micrograph depicting the presence of functional cilia onthe surface of an airway cell differentiated from a human lungprogenitor as described herein. The cilia were observed to beat incoordinated waves, indicating that the airway cell is functional.

FIG. 11A and FIG. 11B is a schematic depicting differentiationpossibilities for Nkx2.1+, Tuj1−, Pax8− cells. Without wishing to bebound by theory, FIG. 11A and FIG. 11B show two possible mechanisms bywhich downstream human lung progenitors can be differentiated fromNkx2.1+, Tuj1−, Pax8− cells.

FIG. 12 is a schematic diagram depicting an exemplary step-wise airwayprogenitor differentiation protocol from iPSC to airway progenitors.

FIG. 13 is a schematic diagram depicting a variation of the protocol forproducing definitive endoderm from human iPSC and ESC; the protocolpermits production of definitive endoderm with high efficiency andconsistency.

FIG. 14A and FIG. 14B show unbiased chemical screening to enhance lungdifferentiation from anterior endoderm. FIG. 14A shows a schematicdiagram depicting the chemical screening platform. FIG. 14B is a tableshowing the lead compounds that facilitated the production of Nkx2.1+lung progenitor cells.

DETAILED DESCRIPTION

The compositions and methods described herein are related, in part, tothe discovery of a method for making isolated human lung progenitorcells from embryonic stem (ES) cells or induced pluripotent stem cells(iPSCs). The presence and isolation of adult progenitor cells from lungtissue is controversial and has achieved only limited success, thereforethe methods and compositions described herein have the advantage thatisolated human lung progenitor cells can be produced in quantitiesuseful for screening assays or treatment of lung diseases/disorders orlung injury. Further, the cell compositions provided herein have notbeen previously isolated and/or cultured from human lung tissue.

Definitions

As used herein the term “human stem cell” refers to a human cell thatcan self-renew and differentiate to at least one cell type. The term“human stem cell” encompasses human stem cell lines, human-derived iPScells, human embryonic stem cells, human pluripotent cells, humanmultipotent stem cells or human adult stem cells.

As used herein, the term “multipotent” refers to the ability of a cellto differentiate into a plurality of different phenotypes. Multipotentcells can generally only differentiate into cells of a single germ layerlineage. This is in contrast to pluripotent cells which can, bydefinition, differentiate into cells of all three germ layers.Pluripotent cells are characterized primarily by their ability todifferentiate to all three germ layers, using, for example, a nude mouseteratoma formation assay. Pluripotency is also evidenced by theexpression of embryonic stem (ES) cell markers, although the preferredtest for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. A pluripotentcell typically has the potential to divide in vitro for a long period oftime, e.g., greater than one year or more than 30 passages.

As used herein, the term “human lung progenitor cell” is a general termthat refers to any progenitor cell that is committed to the pulmonarylineage and also retains the ability to self-renew. One of the firststeps in commitment to the lung lineage is the appearance of the stemcell marker Nkx2.1, however the Nkx2.1 marker can also be detected incells of the thyroid and brain lineage. Therefore, for the purposes ofthis description, the first progenitor cells committed to the lunglineage and encompassed by the term “human lung progenitor cell” arethose cells that express Nkx2.1, but lack Tuj1 and Pax8 cell surfacemarkers. Any progenitor cell that can be differentiated from the Nkx2.1positive, Tuj1 negative, Pax8 negative cell and that retains the abilityto self-renew is also encompassed by the term “human lung progenitorcell.” Other examples of human lung progenitor cells described hereininclude, but are not limited to, Nkx2.1 positive, Sox2 positive proximalmultipotent airway progenitor cells; Nkx2.1 positive, Sox9 positivedistal multipotent lung progenitor cells; and Nkx2.1 positive, p63positive basal airway stem cells. In some embodiments, the human lungprogenitor cells are differentiated into an airway basal stem cell, aciliated cell, a Clara cell, a mucin secreting goblet cell, a type Ipneumocyte, a type II pneumocyte or a neuroendocrine cell when placedunder selected differentiation conditions. A human lung progenitor cellis not a tumor cell or a cancer cell. In one aspect, a human lungprogenitor cell is not derived from an embryo or from an embryonic stemcell or other cell derived in culture from an embryo. In someembodiments, the human lung progenitor cells are differentiated from anautologous cell or from a non-autologous cell. In one embodiment, thehuman lung progenitor cell is not genetically modified or derived from agenetically modified cell.

As used herein, the term “distal multipotent lung progenitor cell”refers to an Nkx2.1 positive, Sox9 positive cell that can differentiateinto all types of epithelial cells of the lung including, but notlimited to, an airway basal stem cell, a ciliated cell, a Clara cell, aneuroendocrine cell, a squamous epithelial cell, a type I pneumocyte, atype II pneumocyte, a bronchioalveolar stem cell (BASC), lungprogenitors located in the bronchioalveolar duct junction (BADJ) withinthe terminal bronchioles, progenitor cells for type I and type IIpneumocytes, and/or a migratory CK14+ cell for both airway epithelialcells and alveolar cells that respond to lung injury (e.g., fluinfection).

As used herein, the term “proximal airway multipotent progenitor” refersto an Nkx2.1 positive, Sox2 positive cell that can differentiate into anairway basal stem cell, a ciliated cell, a Clara cell, a neuroendocrinecell, a squamous epithelial cell, or a migratory CK14+ cell that movesto a bronchioalveolar location for alveolar cell differentiation after alung injury.

By the term “differentiated cell” is meant any primary cell that is not,in its native form, pluripotent as that term is defined herein. Those ofordinary skill in the art recognize that there is a spectrum ofdifferentiation from totipotent or pluripotent cells at one end to fullydifferentiated cells that do not have the normal capacity to naturallydifferentiate to any other phenotype. Thus, a pluripotent cell isdifferentiated relative to a totipotent cell, and a multipotent cell isdifferentiated relative to a pluripotent cell. In some embodiments, theterm “differentiated cell” also refers to a cell of a more specializedcell type derived from a cell of a less specialized cell type (e.g.,from an undifferentiated cell or a reprogrammed cell) where the cell hasundergone a cellular differentiation process.

As used herein, the term “positive for” when referring to a cellpositive for a marker (e.g., Nkx2.1 positive) means that a cell surfacemarker is detectable above background levels on the cell usingimmunofluorescence microscopy or flow cytometry methods, such asfluorescence activated cell sorting (FACS). Alternatively, the terms“positive for” or “expresses a marker” means that expression of mRNAencoding a cell surface or intracellular marker is detectable abovebackground levels using RT-PCR. The expression level of a cell surfacemarker or intracellular marker can be compared to the expression levelobtained from a negative control (i.e., cells known to lack the marker)or by isotype controls (i.e., a control antibody that has no relevantspecificity and only binds non-specifically to cell proteins, lipids orcarbohydrates). Thus, a cell that “expresses” a marker (or is “positivefor a marker”) has an expression level detectable above the expressionlevel determined for the negative control for that marker.

As used herein, the term “negative for” when referring to a cellnegative for a marker (or the term “does not express”) means that a cellsurface marker cannot be detected above background levels on the cellusing immunofluorescence microscopy or flow cytometry methods, such asfluorescence activated cell sorting (FACS). Alternatively, the terms“negative” or “does not express” means that expression of the mRNA foran intracellular marker or cell surface marker cannot be detected abovebackground levels using RT-PCR. The expression level of a cell surfacemarker or intracellular marker can be compared to the expression levelobtained from a negative control (i.e., cells known to lack the marker)or by isotype controls (i.e., a control antibody that has no relevantspecificity and only binds non-specifically to cell proteins, lipids orcarbohydrates). Thus, a cell that “does not express” a marker appearssimilar to the negative control for that marker.

As used herein, the phrase “cell is proliferative” refers to the abilityof a stem cell to self-renew. Self-renewal can occur by either of twomajor mechanisms. Stem cells can divide asymmetrically, with onedaughter retaining the stem state and the other daughter expressing somedistinct other specific function and phenotype. Alternatively, some ofthe stem cells in a population can divide symmetrically into two stems,thus maintaining some stem cells in the population as a whole, whileother cells in the population give rise to differentiated progeny only.

As used herein, the term “capacity to differentiate” refers to theability of a stem cell, progenitor cell, pluripotent cell, ormultipotent cell to differentiate into a subset of more differentiatedcells. The term “capacity to differentiate” does not encompass movingbackwards along the differentiation spectrum such that a cell isproduced that comprises a greater differentiation capacity than theparent cell. That is, the term “capacity to differentiate” does notencompass re-programming methods to shift cells to a less differentiatedstate.

In the context of cell ontogeny, the term “differentiate”, or“differentiating” is a relative term that indicates a “differentiatedcell” is a cell that has progressed further down the developmentalpathway than its precursor cell. Thus in some embodiments, areprogrammed cell as this term is defined herein, can differentiate tolineage-restricted precursor cells (such as a human lung progenitorcell), which in turn can differentiate into other types of precursorcells further down the pathway (such as a tissue specific precursor, forexample, a proximal airway multipotent progenitor cell), and then to anend-stage differentiated cell, which plays a characteristic role in acertain tissue type, and may or may not retain the capacity toproliferate further.

As used herein, the terms “dedifferentiation” or “reprogramming” or“retrodifferentiation” refer to the process that generates a cell thatre-expresses a more stem cell phenotype or a less differentiatedphenotype than the cell from which it is derived. For example, amultipotent cell can be dedifferentiated to a pluripotent cell. That is,dedifferentiation shifts a cell backward along the differentiationspectrum of totipotent cells to fully differentiated cells. Typically,reversal of the differentiation phenotype of a cell requires artificialmanipulation of the cell, for example, by expressing stem cell-specificmRNA and/or proteins. Reprogramming is not typically observed undernative conditions in vivo or in vitro.

As used herein, the term “somatic cell” refers to any cell other than agerm cell, a cell present in or obtained from a pre-implantation embryo,or a cell resulting from proliferation of such a cell in vitro. Statedanother way, a somatic cell refers to any cells forming the body of anorganism, as opposed to germline cells. Every cell type in the mammalianbody—apart from the sperm and ova, the cells from which they are made(gametocytes) and undifferentiated stem cells—is a somatic cell:internal organs, skin, bones, blood, and connective tissue are allsubstantially made up of somatic cells. In some embodiments the somaticcell is a “non-embryonic somatic cell”, by which is meant a somatic cellthat is not present in or obtained from an embryo and does not resultfrom proliferation of such a cell in vitro. In some embodiments thesomatic cell is an “adult somatic cell”, by which is meant a cell thatis present in or obtained from an organism other than an embryo or afetus or results from proliferation of such a cell in vitro. Unlessotherwise indicated the methods for reprogramming a differentiated cell(e.g., to generate an iPSC) can be performed both in vivo and in vitro(where in vivo is practiced when a differentiated cell is present withina subject, and where in vitro is practiced using an isolateddifferentiated cell maintained in culture).

As used herein, the term “adult cell” refers to a cell found throughoutthe body after embryonic development.

The term “isolated cell” as used herein refers to a cell that has beenremoved from an organism in which it was originally found, or adescendant of such a cell. Optionally the cell has been cultured invitro, e.g., in the presence of other cells. Optionally the cell islater introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells as used herein refers to a population of cells that has beenremoved and separated from a mixed or heterogeneous population of cells.In some embodiments, an isolated population is a substantially purepopulation of cells as compared to the heterogeneous population fromwhich the cells were isolated or enriched. In some embodiments, theisolated population is an isolated population of human lung progenitorcells, e.g., a substantially pure population of human lung progenitorcells as compared to a heterogeneous population of cells comprisinghuman lung progenitor cells and cells from which the human lungprogenitor cells were derived.

The term “substantially pure,” with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,preferably at least about 85%, more preferably at least about 90%, andmost preferably at least about 95% pure, with respect to the cellsmaking up a total cell population. That is, the terms “substantiallypure” or “essentially purified,” with regard to a population of lungprogenitor cells, refers to a population of cells that contain fewerthan about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, mostpreferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, ofcells that are not lung progenitor cells as defined by the terms herein.

The terms “enriching” or “enriched” are used interchangeably herein andmean that the yield (fraction) of cells of one type, such as human lungprogenitor cell compositions and cells for use in the methods describedherein, is increased by at least 10%, by at least 15%, by at least 20%,by at least 25%, by at least 30%, by at least 35%, by at least 40%, byat least 45%, by at least 50%, by at least 55%, by at least 60%, by atleast 65%, by at least 70%, or by at least 75%, over the fraction ofcells of that type in the starting biological sample, culture, orpreparation.

As used herein, “proliferating” and “proliferation” refer to an increasein the number of cells in a population (growth) by means of celldivision. Cell proliferation is generally understood to result from thecoordinated activation of multiple signal transduction pathways inresponse to the environment, including growth factors and othermitogens. Cell proliferation can also be promoted by release from theactions of intra- or extracellular signals and mechanisms that block ornegatively affect cell proliferation.

The terms “renewal” or “self-renewal” or “proliferation” are usedinterchangeably herein, and refers to a process of a cell making morecopies of itself (e.g. duplication) of the cell. In some embodiments,lung progenitor cells are capable of renewal of themselves by dividinginto the same undifferentiated cells (e.g. as determined by measuringthe presence of absence of one or more cell surface markers) over longperiods, and/or many months to years. In some instances, proliferationrefers to the expansion of lung progenitor cells by the repeateddivision of single cells into two identical daughter cells.

The term “separation” or “selection” as used herein refers to isolatingdifferent cell types into one or more populations and collecting theisolated population as a target cell population which is enriched in aspecific target stem cell population. Selection can be performed usingpositive selection, whereby a target enriched cell population isretained, or negative selection, whereby non-target cell types arediscarded (thereby enriching for desired target cell types in theremaining cell population).

The term “positive selection” as used herein refers to selection of adesired cell type by retaining the cells of interest. In someembodiments, positive selection involves the use of an agent to assistin retaining the cells of interest, e.g., use of a positive selectionagent such as an antibody which has specific binding affinity for asurface antigen on the desired or target cell. In some embodiments,positive selection can occur in the absence of a positive selectionagent, e.g., in a “touch-free” or closed system, for example, wherepositive selection of a target cell type is based on any of cell size,density and/or morphology of the target cell type.

The term “negative selection” as used herein refers to selection ofundesired or non-target stem cells for depletion or discarding, therebyretaining (and thus enriching) the desired target cell type. In someembodiments, negative selection involves the use of an agent to assistin selecting undesirable cells for discarding, e.g., use of a negativeselection agent such as a monoclonal antibody which has specific bindingaffinity for a surface antigen on unwanted or non-target cells. In someembodiments, negative selection does not involve a negative selectionagent. In some embodiments, negative selection can occur in the absenceof a negative selection agent, e.g., in a “touch-free” or closed system,for example, where negative selection of an undesired (non-target) celltype to be discarded is based on any of cell size, density and/ormorphology of the undesired (non-target) cell type.

The term “marker” as used herein is used to describe the characteristicsand/or phenotype of a cell. Markers can be used for selection of cellscomprising characteristics of interest and can vary with specific cells.Markers are characteristics, whether morphological, functional orbiochemical (enzymatic) characteristics of the cell of a particular celltype, or molecules expressed by the cell type. In one aspect, suchmarkers are proteins. Such proteins can possess an epitope forantibodies or other binding molecules available in the art. However, amarker can consist of any molecule found in a cell including, but notlimited to, proteins (peptides and polypeptides), lipids,polysaccharides, nucleic acids and steroids. Examples of morphologicalcharacteristics or traits include, but are not limited to, shape, size,and nuclear to cytoplasmic ratio. Examples of functional characteristicsor traits include, but are not limited to, the ability to adhere toparticular substrates, ability to incorporate or exclude particulardyes, ability to migrate under particular conditions, and the ability todifferentiate along particular lineages. Markers can be detected by anymethod available to one of skill in the art. Markers can also be theabsence of a morphological characteristic or absence of proteins, lipidsetc. Markers can be a combination of a panel of unique characteristicsof the presence and/or absence of polypeptides and other morphologicalcharacteristics. In one embodiment, the marker is a cell surface marker.Exemplary cell surface markers expressed on lung progenitor cellsinclude, but are not limited to, Sox2, Sox9, p63, FoxP2, ETV4/5, FoxA2,Nkx2.1, Gata6, ID2, CK5, NGFR, FoxJ1, CCSP, Scgb3a2, Muc5ac, T1a, Spc,and Scgn. In some embodiments, the absence of a cell surface marker canbe used to distinguish a lung progenitor cell from a cell of anotherlineage (e.g., a thyroid or brain lineage). Exemplary cell surfacemarkers that are absent on lung progenitor cells or differentiated lungcells include, but are not limited to, Tuj1, and Pax8. One of skill inthe art will recognize that a cell surface marker can be present at aparticular point in development or in a particular lung progenitor celltype. For example, Sox2 is expressed in progenitor cells of the anteriorendoderm, is not expressed in more differentiated lung progenitors, suchas distal multipotent lung progenitors, and then is reactivated in cellssuch as airway progenitors as differentiation of the progenitorsprogresses. Thus, a cell surface marker can be used in combination witha positive selection strategy for certain lung progenitors and also usedas in combination with a negative selection strategy for other lungprogenitors, depending on the particular differentiation stage of thedesired lung progenitor to be selected.

As used herein, the term “scaffold” refers to a structure, comprising abiocompatible material, that provides a surface suitable for adherenceand proliferation of cells. A scaffold can further provide mechanicalstability and support. A scaffold can be in a particular shape or formso as to influence or delimit a three-dimensional shape or form assumedby a population of proliferating cells. Such shapes or forms include,but are not limited to, films (e.g. a form with two-dimensionssubstantially greater than the third dimension), ribbons, cords, sheets,flat discs, cylinders, spheres, 3-dimensional amorphous shapes, etc.

As used herein, the term “implantable in a subject” refers to anynon-living (e.g., acellular) implantable structure that uponimplantation does not generate an appreciable immune response in thehost organism. Thus, an implantable structure should not for example, beor contain an irritant, or contain LPS etc.

As used herein, the term “biodegradable” refers to the ability of ascaffold to degrade under physiological conditions, for example underconditions that do not adversely affect cell viability of the deliveredcells or cells in vivo. Such biodegradable scaffolds will preferably notbe or contain an irritant or an allergen that can cause a systemicreaction in the subject to which the composition has been implanted. Insome embodiments, biodegradable means that the scaffold can bemetabolized and the metabolites cleared from the subject byphysiological excretion mechanisms (e.g., urine, feces, liverdetoxification etc.).

As used herein, the term “treating” includes reducing or alleviating atleast one adverse effect or symptom of a condition, disease or disorder.For example, the term “treating” and “treatment” refers to administeringto a subject an effective amount of a composition, e.g., an effectiveamount of a composition comprising a population of lung progenitor cellsso that the subject has a reduction in at least one symptom of thedisease or an improvement in the disease, for example, beneficial ordesired clinical results. For purposes of this disclosure, beneficial ordesired clinical results include, but are not limited to, alleviation ofone or more symptoms, diminishment of extent of disease, diseasestabilization (e.g., not worsening), delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. In some embodiments, treating can refer to prolongingsurvival as compared to expected survival if not receiving treatment.Thus, one of skill in the art realizes that a treatment can improve thedisease condition, but may not be a complete cure for the disease. Insome embodiments, treatment can include prophylaxis. However, inalternative embodiments, treatment does not include prophylaxis.

“Treatment” of a lung disorder, a lung disease, or a lung injury (e.g.,acute lung injury) as referred to herein refers to therapeuticintervention that stabilizes or improves the function of the lung or theairway. That is, “treatment” is oriented to the function of therespiratory tract. A therapeutic approach that stabilizes or improvesthe function of the lung or the airway by at least 10%, and preferablyby at least 20%, 30%, 40%, 50%, 75%, 90%, 100% or more, e.g., 2-fold,5-fold, 10-fold or more, up to and including full function, relative tosuch function prior to such therapy is considered effective treatment.Effective treatment need not cure or directly impact the underlyingcause of the lung disease or disorder to be considered effectivetreatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike. A pharmaceutically acceptable carrier will not promote the raisingof an immune response to an agent with which it is admixed, unless sodesired. The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art and need not be limited based on formulation. Typically suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient. The therapeutic composition ofthe present invention can include pharmaceutically acceptable salts ofthe components therein. Pharmaceutically acceptable salts include theacid addition salts (formed with the free amino groups of thepolypeptide) that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,tartaric, mandelic and the like. Salts formed with the free carboxylgroups can also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium or ferric hydroxides, and suchorganic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine and the like. Physiologically tolerable carriers arewell known in the art. Exemplary liquid carriers are sterile aqueoussolutions that contain no materials in addition to the activeingredients and water, or contain a buffer such as sodium phosphate atphysiological pH value, physiological saline or both, such asphosphate-buffered saline. Still further, aqueous carriers can containmore than one buffer salt, as well as salts such as sodium and potassiumchlorides, dextrose, polyethylene glycol and other solutes. Liquidcompositions can also contain liquid phases in addition to and to theexclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions. The amount of an active agent used with the methods describedherein that will be effective in the treatment of a particular disorderor condition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or symptoms thereof, refers to a reduction in thelikelihood that an individual will develop a disease or disorder, e.g.,a lung disorder. The likelihood of developing a disease or disorder isreduced, for example, when an individual having one or more risk factorsfor a disease or disorder either fails to develop the disorder ordevelops such disease or disorder at a later time or with less severity,statistically speaking, relative to a population having the same riskfactors and not receiving treatment as described herein. The failure todevelop symptoms of a disease, or the development of reduced (e.g., byat least 10% on a clinically accepted scale for that disease ordisorder) or delayed (e.g., by days, weeks, months or years) symptoms isconsidered effective prevention.

As used herein, the term “induced to differentiate” refers to achemical/biological treatment, a physical environment or a geneticmodification that is conducive to the formation of more differentiatedcells (e.g., human lung progenitor cells or cells having an airwayphenotype) from pluripotent or multipotent stem cells (e.g., anteriorforegut endoderm cells). Differentiation can be assessed by theappearance of distinct cell-type specific markers or by the loss of stemcell specific markers, or both.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Embryonic Stem Cells

Stem cells are cells that retain the ability to renew themselves throughmitotic cell division and can differentiate into a diverse range ofspecialized cell types. Three broad types of mammalian stem cellsinclude: embryonic stem (ES) cells that are found in blastocysts,induced pluripotent stem cells (iPSCs) that are reprogrammed fromsomatic cells, and adult stem cells that are found in adult tissues. Ina developing embryo, stem cells can differentiate into all of thespecialized embryonic tissues. In adult organisms, stem cells andprogenitor cells act as a repair system for the body, replenishingspecialized cells, but also maintain the normal turnover of regenerativeorgans, such as blood, skin or intestinal tissues. Pluripotent stemcells can differentiate into cells derived from any of the three germlayers.

Stem cells are classified by their developmental potential as: (1)totipotent, which is able to give rise to all embryonic andextraembryonic cell types; (2) pluripotent, which is able to give riseto all embryonic cell types, i.e., endoderm, mesoderm, and ectoderm; (3)multipotent, which is able to give rise to a subset of cell lineages,but all within a particular tissue, organ, or physiological system (forexample, multipotent distal lung progenitor cells can produce progenythat include multipotent distal lung progenitor cells (self-renewal) andthe cell types and elements (e.g., basal cells, ciliated cells, Claracells and goblet cells) that are normal components of the airway); (4)oligopotent, which is able to give rise to a more restricted subset ofcell lineages than multipotent stem cells; and (5) unipotent, which isable to give rise to a single cell lineage (e.g., spermatogenic stemcells).

Provided herein are methods of generating human lung progenitor cellsfrom both embryonic stem cells and induced pluripotent stem cells. Inone embodiment, the methods provided herein relate to generation ofhuman lung progenitor cells from embryonic stem cells. Alternatively, insome embodiments, the methods provided herein do not encompassgeneration of human lung progenitor cells from embryonic stem cells orany other cells of human embryonic origin.

Embryonic stem cells and methods of their retrieval are well known inthe art and are described, for example, in Trounson A O (Reprod FertilDev (2001) 13: 523), Roach M L (Methods Mol Biol (2002) 185: 1), andSmith A G (Annu Rev Cell Dev Biol (2001) 17:435). The term “embryonicstem cell” is used to refer to the pluripotent stem cells of the innercell mass of the embryonic blastocyst (see e.g., U.S. Pat. Nos.5,843,780, 6,200,806). Such cells can similarly be obtained from theinner cell mass of blastocysts derived from somatic cell nucleartransfer (see, for example, U.S. Pat. Nos. 5,945,577, 5,994,619,6,235,970). The distinguishing characteristics of an embryonic stem celldefine an embryonic stem cell phenotype. Accordingly, a cell has thephenotype of an embryonic stem cell if it possesses one or more of theunique characteristics of an embryonic stem cell such that that cell canbe distinguished from other cells. Exemplary distinguishing embryonicstem cell characteristics include, without limitation, gene expressionprofile, proliferative capacity, differentiation capacity, karyotype,responsiveness to particular culture conditions, and the like.

Cells derived from embryonic sources can include embryonic stem cells orstem cell lines obtained from a stem cell bank or other recognizeddepository institution. Other means of producing stem cell lines includemethods comprising the use of a blastomere cell from an early stageembryo prior to formation of the blastocyst (at around the 8-cellstage). Such techniques correspond to the pre-implantation geneticdiagnosis technique routinely practiced in assisted reproductionclinics. The single blastomere cell is co-cultured with establishedES-cell lines and then separated from them to form fully competent EScell lines.

Embryonic stem cells are considered to be undifferentiated when theyhave not committed to a specific differentiation lineage. Such cellsdisplay morphological characteristics that distinguish them fromdifferentiated cells of embryo or adult origin. Undifferentiatedembryonic stem (ES) cells are easily recognized by those skilled in theart, and typically appear in the two dimensions of a microscopic view incolonies of cells with high nuclear/cytoplasmic ratios and prominentnucleoli. In some embodiments, the human lung progenitor cells describedherein are not derived from embryonic stem cells or any other cells ofembryonic origin.

Adult stem cells are stem cells, which are derived from tissues of apost-natal or post-neonatal organism or from an adult organism are alsoknown in the art. An adult stem cell is structurally distinct from anembryonic stem cell not only in markers it does or does not expressrelative to an embryonic stem cell, but also by the presence ofepigenetic differences, e.g. differences in DNA methylation patterns.

Induced Pluripotent Stem Cells (iPSCs)

In some embodiments, the human lung progenitor cells described hereinare derived from isolated pluripotent stem cells. An advantage of usingiPSCs is that the cells can be derived from the same subject to whichthe human lung progenitor cells are to be administered. That is, asomatic cell can be obtained from a subject, reprogrammed to an inducedpluripotent stem cell, and then re-differentiated into a human lungprogenitor cell to be administered to the subject (e.g., autologouscells). Since the lung progenitors are essentially derived from anautologous source, the risk of engraftment rejection or allergicresponses is reduced compared to the use of cells from another subjector group of subjects. In some embodiments, the lung progenitors arederived from non-autologous sources. In addition, the use of iPSCsnegates the need for cells obtained from an embryonic source. Thus, inone embodiment, the stem cells used in the disclosed methods are notembryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to induced pluripotent stem cells. Exemplary methods areknown to those of skill in the art and are described briefly hereinbelow.

As used herein, the term “reprogramming” refers to a process that altersor reverses the differentiation state of a differentiated cell (e.g., asomatic cell). Stated another way, reprogramming refers to a process ofdriving the differentiation of a cell backwards to a moreundifferentiated or more primitive type of cell. It should be noted thatplacing many primary cells in culture can lead to some loss of fullydifferentiated characteristics. Thus, simply culturing such cellsincluded in the term differentiated cells does not render these cellsnon-differentiated cells (e.g., undifferentiated cells) or pluripotentcells. The transition of a differentiated cell to pluripotency requiresa reprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. In some embodiments,reprogramming encompasses complete reversion of the differentiationstate of a differentiated cell (e.g., a somatic cell) to a pluripotentstate or a multipotent state. In some embodiments, reprogrammingencompasses complete or partial reversion of the differentiation stateof a differentiated cell (e.g., a somatic cell) to an undifferentiatedcell (e.g., an embryonic-like cell). Reprogramming can result inexpression of particular genes by the cells, the expression of whichfurther contributes to reprogramming. In certain embodiments describedherein, reprogramming of a differentiated cell (e.g., a somatic cell)causes the differentiated cell to assume an undifferentiated state(e.g., is an undifferentiated cell). The resulting cells are referred toas “reprogrammed cells,” or “induced pluripotent stem cells (iPSCs oriPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a hematopoietic stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some embodiments.

The specific approach or method used to generate pluripotent stem cellsfrom somatic cells (broadly referred to as “reprogramming”) is notcritical to the claimed invention. Thus, any method that re-programs asomatic cell to the pluripotent phenotype would be appropriate for usein the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been describedinduced pluripotent stem cells. Yamanaka and Takahashi converted mousesomatic cells to ES cell-like cells with expanded developmentalpotential by the direct transduction of Oct4, Sox2, Klf4, and c-Myc(Takahashi and Yamanaka, 2006). iPSCs resemble ES cells as they restorethe pluripotency-associated transcriptional circuitry and much of theepigenetic landscape. In addition, mouse iPSCs satisfy all the standardassays for pluripotency: specifically, in vitro differentiation intocell types of the three germ layers, teratoma formation, contribution tochimeras, germline transmission (Maherali and Hochedlinger, 2008), andtetraploid complementation (Woltjen et al., 2009).

Subsequent studies have shown that human iPS cells can be obtained usingsimilar transduction methods (Lowry et al., 2008; Park et al., 2008;Takahashi et al., 2007; Yu et al., 2007b), and the transcription factortrio, OCT4, SOX2, and NANOG, has been established as the core set oftranscription factors that govern pluripotency (Jaenisch and Young,2008). The production of iPS cells can be achieved by the introductionof nucleic acid sequences encoding stem cell-associated genes into anadult, somatic cell, historically using viral vectors.

iPS cells can be generated or derived from terminally differentiatedsomatic cells, as well as from adult stem cells, or somatic stem cells.That is, a non-pluripotent progenitor cell can be rendered pluripotentor multipotent by reprogramming. In such instances, it may not benecessary to include as many reprogramming factors as required toreprogram a terminally differentiated cell. Further, reprogramming canbe induced by the non-viral introduction of reprogramming factors, e.g.,by introducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 2010 Nov. 5; 7(5):618-30). Reprogrammingcan be achieved by introducing a combination of nucleic acids encodingstem cell-associated genes including, for example Oct-4 (also known asOct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28. In oneembodiment, reprogramming using the methods and compositions describedherein can further comprise introducing one or more of Oct-3/4, a memberof the Sox family, a member of the Klf family, and a member of the Mycfamily to a somatic cell. In one embodiment, the methods andcompositions described herein further comprise introducing one or moreof each of Oct 4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. Asnoted above, the exact method used for reprogramming is not necessarilycritical to the methods and compositions described herein. However,where cells differentiated from the reprogrammed cells are to be usedin, e.g., human therapy, in one embodiment the reprogramming is noteffected by a method that alters the genome. Thus, in such embodiments,reprogramming is achieved, e.g., without the use of viral or plasmidvectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various small molecules as shown by Shi, Y., et al (2008)Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3:132-135.Thus, an agent or combination of agents that enhance the efficiency orrate of induced pluripotent stem cell production can be used in theproduction of patient-specific or disease-specific iPSCs. Somenon-limiting examples of agents that enhance reprogramming efficiencyinclude soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histonemethyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferaseinhibitors, histone deacetylase (HDAC) inhibitors, valproic acid,5′-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA),vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VPA)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE(2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Otherreprogramming enhancing agents include, for example, dominant negativeforms of the HDACs (e.g., catalytically inactive forms), siRNAinhibitors of the HDACs, and antibodies that specifically bind to theHDACs. Such inhibitors are available, e.g., from BIOMOL International,Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, AtonPharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, andSigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto,Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, a cellthat expresses Oct4 or Nanog is identified as pluripotent. Methods fordetecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses. In someembodiments, detection does not involve only RT-PCR, but also includesdetection of protein markers. Intracellular markers may be bestidentified via RT-PCR, while cell surface markers are readilyidentified, e.g., by immunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate to cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells are introduced to nude mice and histologyand/or immunohistochemistry is performed on a tumor arising from thecells. The growth of a tumor comprising cells from all three germlayers, for example, further indicates that the cells are pluripotentstem cells.

Somatic Cells for Reprogramming:

Somatic cells, as that term is used herein, refer to any cells formingthe body of an organism, excluding germline cells. Every cell type inthe mammalian body—apart from the sperm and ova, the cells from whichthey are made (gametocytes) and undifferentiated stem cells—is adifferentiated somatic cell. For example, internal organs, skin, bones,blood, and connective tissue are all made up of differentiated somaticcells.

Additional somatic cell types for use with the compositions and methodsdescribed herein include: a fibroblast (e.g., a primary fibroblast), amuscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a mammarycell, an hepatocyte and a pancreatic islet cell. In some embodiments,the somatic cell is a primary cell line or is the progeny of a primaryor secondary cell line. In some embodiments, the somatic cell isobtained from a human sample, e.g., a hair follicle, a blood sample, abiopsy (e.g., a skin biopsy or an adipose biopsy), a swab sample (e.g.,an oral swab sample), and is thus a human somatic cell.

Some non-limiting examples of differentiated somatic cells include, butare not limited to, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, immune cells, hepatic, splenic, lung, circulating bloodcells, gastrointestinal, renal, bone marrow, and pancreatic cells. Insome embodiments, a somatic cell can be a primary cell isolated from anysomatic tissue including, but not limited to brain, liver, lung, gut,stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ,bone, etc. Further, the somatic cell can be from any mammalian species,with non-limiting examples including a murine, bovine, simian, porcine,equine, ovine, or human cell. In some embodiments, the somatic cell is ahuman somatic cell.

When reprogrammed cells are used for generation of human lung progenitorcells to be used in the therapeutic treatment of disease, it isdesirable, but not required, to use somatic cells isolated from thepatient being treated. For example, somatic cells involved in diseases,and somatic cells participating in therapeutic treatment of diseases andthe like can be used. In some embodiments, a method for selecting thereprogrammed cells from a heterogeneous population comprisingreprogrammed cells and somatic cells they were derived or generated fromcan be performed by any known means. For example, a drug resistance geneor the like, such as a selectable marker gene can be used to isolate thereprogrammed cells using the selectable marker as an index.

Reprogrammed somatic cells as disclosed herein can express any number ofpluripotent cell markers, including: alkaline phosphatase (AP); ABCG2;stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60;TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; β-III-tubulin; α-smoothmuscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1;zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cellassociated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7;ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14;undifferentiated embryonic cell transcription factor (Utf1); Rex1; p53;G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a;Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4;Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3;CYP25A1; developmental pluripotency-associated 2 (DPPA2); T-celllymphoma breakpoint 1 (Tcl1); DPPA3/Stella; DPPA4; other general markersfor pluripotency, etc. Other markers can include Dnmt3L; Sox15; Stat3;Grb2; β-catenin, and Bmi1. Such cells can also be characterized by thedown-regulation of markers characteristic of the somatic cell from whichthe induced pluripotent stem cell is derived.

Generation of Definitive Endoderm and Anterior Foregut Endoderm

The methods for generating human lung progenitor cells as describedherein begin by first generating definitive endoderm from embryonic stemcells or induced pluripotent stem cells. “Definitive endoderm” comprisesa multipotent cell committed to the endoderm lineage and that can giverise to cells of the gut tube or organs derived from the gut tube. Thedefinitive endoderm is the germ layer that gives rise togastrointestinal organs (e.g., esophagus, stomach, liver, gall bladder,small intestines, pancreas, colon, etc.), respiratory organs (e.g.,alveoli, trachea, bronchi), endocrine glands and organs (e.g.,parathyroid gland, thyroid gland, thymus), auditory system (e.g.,auditory tube, and tympanic cavity), and the urinary system (e.g.,urinary bladder and portions of the urethra). See e.g., Grapin-Bottonand Melton, 2000; Kimelman and Griffin, 2000; Tremblay et al., 2000;Wells and Melton, 1999; Wells and Melton, 2000. The term “definitiveendoderm” does not encompass the separate lineage of cells termedprimitive endoderm, which is responsible for formation ofextra-embryonic tissues.

Formation of definitive endoderm and endoderm cells derived therefrom isan important step for the derivation of cells which make up terminallydifferentiated tissues and/or organs derived from the definitiveendoderm lineage, such as the human lung progenitor cells as describedherein.

Methods for deriving definitive endoderm from embryonic stem cells orinduced pluripotent stem cells are known in the art (e.g., U.S. Pat.Nos. 7,993,916; 7,695,963; 7,541,185; US2009/0298178; US2010/0272695;Sherwood et al., Mechanisms of Development (2011) 128:387-400; D'AmourK. et al., Nature Biotechnology (2005) 23:1534-1541; Turovets, N. etal., Differentiation (2011) 81(5):292-298; Kim, P T. et al., PLoS One(2010) 5(11):e14146). In one embodiment, definitive endoderm is producedby contacting an IPSC or ESC with a definitive endoderm medium includinge.g., B27, Activin A and ZSTK474. In one embodiment, the definitiveendoderm medium comprises 1-5% B27 (e.g., 2%), 10-40 ng/mL Activin A(e.g., 20 ng/mL), and 0.2-0.5 μM ZSTK474 and is useful for generatingdefinitive endoderm in cell lines that have been tested to have lowefficiency of definitive endoderm generation or are suspected of havinga low efficiency of definitive endoderm generation.

Differentiation of embryonic stem cells or induced pluripotent stemcells to definitive endoderm can be monitored by determining theexpression of cell surface markers characteristic of definitiveendoderm. In some embodiments, the expression of definitive endodermmarkers is determined by detecting the presence or absence of themarker. Alternatively, the expression of certain markers can bedetermined by measuring the level at which the marker is present in thecells of the cell culture or cell population. Such measurements ofmarker expression can be either qualitative or quantitative.

In one embodiment, quantitative PCR (Q-PCR) is used to quantify theexpression of markers on the definitive endoderm. Methods of performingQ-PCR are well known in the art. In alternative embodiments, expressionof a marker gene product is detected using antibodies specific for thecell marker. In certain embodiments, the expression of marker genescharacteristic of definitive endoderm as well as the lack of significantexpression of marker genes characteristic of the cells from which theyare derived (e.g., ES cells or iPSCs) and other cell types isdetermined.

In one embodiment, a marker of definitive endoderm is the SOX17 gene.Other markers of definitive endoderm include, but are not limited to,MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. Insome embodiments, the expression of both SOX17 and SOX7 is monitored. Inother embodiments, expression of the SOX17 marker gene and the OCT4marker gene, which is characteristic of ES cells, is monitored.Additionally, because definitive endoderm cells express the SOX17 markergene at a level higher than that of the AFP, SPARC or Thrombomodulinmarker genes, the expression of these genes can also be monitored.Another marker of definitive endoderm is the CXCR4 gene, which encodes acell surface chemokine receptor whose ligand is the chemoattractantSDF-1. In one embodiment, the efficiency of definitive endodermproduction can be determined by costaining for FOXA2/SOX17 or by FACSanalysis with cKit/CXCR4 or cKit/EpCAM combination.

Once generation of definitive endoderm has been achieved, the next stepis to differentiate the definitive endoderm cells to anterior foregutendoderm cells, which is the region that comprises the cells destined tobecome lung and thyroid cells. This process is also referred to hereinas “anteriorization” of definitive endoderm. As used herein, “foregutendoderm” refers to cells of the anterior portion of the gut tube andencompasses cells of the foregut/midgut junction. It will be recognizedby one of skill in the art that ESCs or iPSCs can also be differentiateddirectly to anterior foregut endoderm cells without requiring anintermediate step of generating definitive endoderm. The differentiationmethods described herein for generating lung progenitor cells begin fromanterior foregut endoderm cells, and thus the method of making suchanterior foregut endoderm cells is not critical and is not limited tothe methods for generating anterior foregut endoderm that are describedherein; any method that provides anterior foregut endoderm can be usedto provide the starting material for preparation of lung progenitorcells, as disclosed herein.

Methods for generating anterior foregut endoderm from definitiveendoderm are known in the art (see e.g., WO2010/136583, WO2011/139628;Green, M D et al., Nature Biotechnology (2011) 29:267-27; Morrison etal, (2008), Cell Stem Cell, 3: 355-356; Goss A M et al., DevelopmentalCell (2009) 17(2):290-298; Livigni A et al., Current Protocols in StemCell Biology (2009) 10:1G.3.1-1G.3.10).

In one embodiment, the production of anterior foregut endoderm isconfirmed by the activation of an anterior foregut endoderm specificmarker, such as the marker Hex. Hex is a homeobox-containingtranscriptional repressor that is one of the earliest markers ofanterior foregut endoderm, and has been shown to suppress posteriorcharacteristics (see e.g., Brickman J M et al., Development (2000)127:2303-2315; Thomas P Q et al., Development (1998) 125:85-94;Zamparini A L et al., Development (2006) 133:3709-3722). The detectionof Hex can be used in combination with other anterior foregut endodermmarkers, such as Cxcr4 (Morrison, G M et al., Cell Stem Cell (2008)3:402-412). Other exemplary markers include, but are not limited to,FoxA2 and Sox2, among others. In one embodiment, the definitive endodermundergoes an anteriorization step comprising treatment with a TGFβagonist (e.g., Activin).

Signaling Pathways for Differentiation

TGF-β signaling pathway modulation: In some embodiments, one or moreTGF-β agonists are used to promote a particular differentiation step ofa pluripotent cell (e.g., during generation of anterior foregutendoderm). In such embodiments, an activating agent specific for TGF-βsignaling can be a TGF-β polypeptide or an active fragment thereof, afusion protein comprising a TGF-β polypeptide or an active fragmentthereof, an agonist antibody to a TGF-β receptor, or a small moleculeagonist of a TGF-β receptor.

In other embodiments, one or more TGF-β antagonists can be used topermit differentiation of a pluripotent cell (e.g., for inducing Nkx2.1expression, the first step towards commitment to lung lineage). In suchembodiments, an antagonist for TGF-β signaling can be a polypeptideinhibitor or a fragment thereof, a dominant negative fusion protein, anantagonist antibody to a TGF-β receptor or a small molecule antagonistof a TGF-β receptor.

The Transforming growth factor beta (TGF-β) signaling pathway isinvolved in many cellular processes in both the adult organism and thedeveloping embryo including cell growth, cell differentiation,apoptosis, cellular homeostasis and other cellular functions. TGF-βsuperfamily ligands bind to a type II receptor, which recruits andphosphorylates a type I receptor. The type I receptor thenphosphorylates receptor-regulated SMADs (R-SMADs) which then bind thecoSMAD SMAD4. R-SMAD/coSMAD complexes accumulate in the nucleus wherethey act as transcription factors and participate in the regulation oftarget gene expression.

TGF-β1 is a prototypic member of a family of cytokines including theTGF-βs, activins, inhibins, bone morphogenetic proteins andMullerian-inhibiting substance. Smad proteins are exemplary downstreamsignal transduction factors in the TGF-beta pathway and therefore, insome embodiments, can be activated directly to effect differentiation toa human lung cell progenitor phenotype (e.g., by treating a cell with anactivator of a Smad protein). Exemplary Smad activators include, but arenot limited to, Smad proteins or functional peptides or fragmentsthereof (e.g., Smad1, Smad5, Smad8), BMP2, BMP4, and Mullerianinhibiting substance (MIS). Activin ligands transduce signals in amanner similar to TGF-β ligands. Activins bind to and activate ALKreceptors, which in turn phosphorylate Smad proteins such as Smad2 andSmad3. The consequent formation of a hetero-Smad complex with Smad4results in the activin-induced regulation of gene transcription.

Some non-limiting examples of small molecule inhibitors of TGF-βreceptors include 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5napththyridine, [3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole,which can be purchased from Calbiochem (San Diego, Calif.). Other smallmolecule inhibitors include, but are not limited to, SB-431542 (seee.g., Halder et al., 2005; Neoplasia 7(5):509-521), SM16 (see e.g., Fu,K et al., 2008; Arteriosclerosis, Thrombosis and Vascular Biology28(4):665), and SB-505124 (see e.g., Dacosta Byfield, S., et al., 2004;Molecular Pharmacology 65:744-52), among others. Additional TGF-βreceptor antagonists are known in the art.

In some embodiments, the dosage range useful for a TGF-β antagonist(e.g., A8301) is between 0.1 and 10 μM, for example between 0.1 and 1μM, between 0.1 and 0.5 μM, between 0.1 and 2 μM, between 0.1 and 3 μM,between 0.1 and 4 μM, between 0.1 and 5 μM, between 0.1 and 6 μM,between 0.1 and 7 μM, between 0.1 and 8 μM, between 0.1 and 9 μM,between 0.5 and 2 μM, between 0.5 and 5 μM, between 1 and 3 μM, between2 and 4 μM, between 2 and 6 μM, between 2 and 7 μM, between 5 and 10 μM,between 6 and 10 μM, between 7 and 10 μM, between 8 and 10 μM, between 9and 10 μM. In some embodiments the TGF-β antagonist is used at a dose ofe.g., at least 0.1 μM, at least 0.2 μM, at least 0.3 μM, at least 0.4μM, at least 0.5 μM, at least 0.6 μM, at least 0.7 μM, at least 0.8 μM,at least 0.9 μM, at least 1 μM, at least 1.2 μM, at least 1.3 μM, atleast 1.4 μM, at least 1.5 μM, at least 1.6 μM, at least 1.7 μM, atleast 1.8 μM, at least 1.9 μM, at least 2 μM, at least 2.5 μM, at least3 μM, at least 3.5 μM, at least 4 μM, at least 4.5 μM, at least 5 μM, atleast 5.5 μM, at least 6 μM, at least 6.5 μM, at least 7 μM, at least7.5 μM, at least 8 μM, at least 8.5 μM, at least 9 μM, at least 9.5 μM,at least 10 μM or more.

BMP Receptor Signaling Pathway Modulation

BMP2 and BMP4 both signal through the type I receptor (ALK3), while BMP7binds to a separate type I receptor (ALK2). See e.g., von Bubnoff A etal., Developmental Biology (2001) 239:1-14; Chen D. et al., GrowthFactors (2004) 22(4):233-241; Sieber C. et al., Cytokine and GrowthFactor Rev. (2009) 20:343-355; and Miyazono K et al., Journal ofBiochemistry (2010) 147(1):35-51.

Typically, BMP2 and BMP4 bind to a BMP receptor I/II complex, leading tophosphorylation of Smads 1/5/8, followed by formation of heterotrimericcomplexes with Smad4. These complexes translocate to the nucleus andactivate expression of target genes (von Bubnoff A et al., DevelopmentalBiology (2001) 239:1-14; Chen D. et al., Growth Factors (2004)22(4):233-241; Sieber C. et al., Cytokine and Growth Factor Rev. (2009)20:343-355; and Miyazono K et al., Journal of Biochemistry (2010)147(1):35-51). Besides Smad1/5/8-mediated transcription, BMP-inducedreceptor complexes can activate the mitogen-activated protein kinase(MAPK) pathway via ERK, JNK, or p38 (Kozawa O et al., Journal ofCellular Biochemistry 84:583-589).

BMP receptor pathway activation: In some embodiments, a BMP agonist isused with the methods described herein for differentiation of a humanlung progenitor cell. In one embodiment, the BMP receptor is a receptorthat signals through the SMAD pathway (e.g., ALK3). In otherembodiments, the BMPs used with the methods described herein are BMP2and/or BMP4.

In one embodiment, one or more BMP agonists are used to promote aparticular differentiation step of a pluripotent cell. In suchembodiments, an activating agent specific for BMP signaling can be a BMPpolypeptide or an active fragment thereof, a fusion protein comprising aBMP polypeptide or an active fragment thereof, an agonist antibody to aBMP receptor, or a small molecule agonist of a BMP receptor.

In some embodiments, the dosage range useful for BMP4 is between 1 and500 nM, for example between 1 and 400 nM, between 1 and 300 nM, between1 and 200 nM, between 1 and 100 nM, between 1 and 50 nM, between 1 and25 nM, between 1 and 10 nM, between 1 and 5 nM, between 1 and 2 nM,between 10 and 300 nM, between 15 and 250 nM, between 20 and 250 nM,between 20 and 200 nM, between 30 and 200 nM, between 40 and 200 nM,between 50 and 200 nM, between 60 and 200 nM, between 70 and 200 nM,between 80 and 200 nM, between 90 and 200 nM, between 100 and 200 nM,between 150 and 200 nM, between 150 nM and 300 nM, between 175 and 300nM, between 200 nM and 300 nM, between 200 nM and 400 nM, between 200 nMand 500 nM.

In some embodiments the dose of BMP4 is e.g., at least 1 nM, at least 2nM, at least 5 nM, at least 10 nM, at least 20 nM, at least 30 nM, atleast 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80nM, at least 90 nM, at least 100 nM, at least 110 nM, at least 120 nM,at least 130 nM, at least 140 nM, at least 150 nM, at least 160 nM, atleast 170 nM, at least 180 nM, at least 190 nM, at least 200 nM, atleast 225 nM, at least 250 nM, at least 275 nM, at least 300 nM, atleast 400 nM, at least 500 nM or more.

In some embodiments, the dosage range useful for BMP7 is between 1 and200 ng/mL, for example between 1 and 100 ng/mL, between 1 and 50 ng/mL,between 1 and 25 ng/mL, between 1 and 10 ng/mL, between 1 and 5 ng/mL,between 1 and 2 ng/mL, between 10 and 200 ng/mL, between 15 and 200ng/mL, between 20 and 200 ng/mL, between 30 and 200 ng/mL, between 40and 200 ng/mL, between 50 and 200 ng/mL, between 60 and 200 ng/mL,between 70 and 200 ng/mL, between 80 and 200 ng/mL, between 90 and 200ng/mL, between 100 and 200 ng/mL, or between 150 and 200 ng/mL.

In some embodiments the dose of BMP7 is e.g., at least 1 ng/mL, at least2 ng/mL, at least 5 ng/mL, at least 10 ng/mL, at least 20 ng/mL, atleast 30 ng/mL, at least 40 ng/mL, at least 50 ng/mL, at least 60 ng/mL,at least 70 ng/mL, at least 80 ng/mL, at least 90 ng/mL, at least 100ng/mL, at least 110 ng/mL, at least 120 ng/mL, at least 130 ng/mL, atleast 140 ng/mL, at least 150 ng/mL, at least 160 ng/mL, at least 170ng/mL, at least 180 ng/mL, at least 190 ng/mL, at least 200 ng/mL, ormore.

BMP receptor pathway inhibition: In some embodiments, a BMP antagonistis used with the methods described herein for differentiation of a humanforegut endoderm cell to a lung progenitor cell. In one embodiment, theBMP antagonist is dorsomorphin.

In one embodiment, one or more BMP receptor pathway antagonists are usedto promote a particular differentiation step of a pluripotent cell. Insuch embodiments, an inhibitor specific for BMP signaling can be apolypeptide or fragment thereof, an shRNA or siRNA directed against aBMP receptor, an antagonist antibody to a BMP receptor, or a smallmolecule antagonist of a BMP receptor.

In some embodiments, the dosage range useful for a BMP pathway inhibitoris between 1 and 500 nM, for example between 1 and 400 nM, between 1 and300 nM, between 1 and 200 nM, between 1 and 100 nM, between 1 and 50 nM,between 1 and 25 nM, between 1 and 10 nM, between 1 and 5 nM, between 1and 2 nM, between 10 and 300 nM, between 15 and 250 nM, between 20 and250 nM, between 20 and 200 nM, between 30 and 200 nM, between 40 and 200nM, between 50 and 200 nM, between 60 and 200 nM, between 70 and 200 nM,between 80 and 200 nM, between 90 and 200 nM, between 100 and 200 nM,between 150 and 200 nM, between 150 nM and 300 nM, between 175 and 300nM, between 200 nM and 300 nM, between 200 nM and 400 nM, between 200 nMand 500 nM.

In some embodiments the dose of the BMP pathway antagonist is e.g., atleast 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 20nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, atleast 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least110 nM, at least 120 nM, at least 130 nM, at least 140 nM, at least 150nM, at least 160 nM, at least 170 nM, at least 180 nM, at least 190 nM,at least 200 nM, at least 225 nM, at least 250 nM, at least 275 nM, atleast 300 nM, at least 400 nM, at least 500 nM or more.

MAPKK/ERK inhibitors: Provided herein are methods for differentiating ahuman lung progenitor cell to a Nkx2.1+, Sox2+ proximal multipotentairway progenitor cell or to an Nkx2.1+, Sox9 distal multipotentprogenitor cell, wherein the methods comprise treatment with a MAPKK/ERKinhibitor.

Mitogen activated protein kinase (MAPK) signaling pathways are involvedin cellular events such as growth, differentiation and stress responses(J. Biol. Chem. (1993) 268, 14553-14556). Four parallel MAPK pathwayshave been identified to date: ERK1/ERK2, JNK, p38 and ERK5. Thesepathways are linear kinase cascades in that MAPKKK phosphorylates andactivates MAPKK, and MAPKK phosphorylates and activates MAPK. To date,seven MAPKK homologs (MEK1, MEK2, MKK3, MKK4/SEK, MEK5, MKK6, and MKK7)and four MAPK families (ERK1/2, JNK, p38, and ERK5) have beenidentified. Activation of these pathways regulates the activity of anumber of substrates through phosphorylation. These substrates include:transcription factors such as TCF, c-myc, ATF2 and the AP-1 components,fos and Jun; cell surface components EGF-R; cytosolic componentsincluding PHAS-T, p90^(rsk), cPLA₂ and c-Raf-1; and cytoskeletoncomponents such as tau and MAP2. MAPK signaling cascades are involved incontrolling cellular processes including proliferation, differentiation,apoptosis, and stress responses.

MEK occupies a strategic downstream position in the Mek/Erk pathwaycatalyzing the phosphorylation of its MAPK substrates, ERK1 and ERK2.Anderson et al. Nature 1990, v. 343, pp. 651-653. In the ERK pathway,MAPKK corresponds with MEK (MAP kinase ERK Kinase) and the MAPKcorresponds with ERK (Extracellular Regulated Kinase).

Some non-limiting examples of MAPK and/or ERK pathway inhibitors includeSL327, U0126, SP600125, PD98059, SB203580, and CAY10561. Additional MAPKand/or ERK pathway inhibitors that can be used with the methodsdescribed herein are known to those of skill in the art.

In some embodiments, the dosage range useful for a MAPKK/ERK antagonist(e.g., PD98059) is between 0.1 and 5 μM, for example, between 0.1 and 4μM, between 0.1 and 3 μM, between 0.1 and 2 μM, between 0.1 and 1 μM,between 0.1 and 0.5 μM, between 0.5 and 3 μM, between 0.5 and 2 μM,between 0.5 and 1 μM, between 1 and 2 μM, between 1.5 and 2 μM, between1 and 1.5 μM, between 2 and 5 μM, between 3 and 5 μM, between 4 and 5μM.

In some embodiments, the dose of a MAPKK/ERK antagonist is e.g., atleast 0.1 μM, at least 0.5 μM, at least 1 μM, at least 1.1 μM, at least1.2 μM, at least 1.3 μM, at least 1.4 μM, at least 1.5 μM, at least 1.6μM, at least 1.7 μM, at least 1.8 μM, at least 1.9 μM, at least 2 μM, atleast 2.5 μM, at least 3 μM, at least 4 μM, at least 5 μM or more.

FGF activation: Fibroblast growth factors, or FGFs, are a family ofgrowth factors that play a role in angiogenesis, wound healing, andembryonic development. FGFs and functional fragments or analogs thereofare useful for differentiating human lung progenitor cells to e.g.,proximal multipotent airway progenitor cells, distal multipotent lungprogenitor cells, and multipotent airway basal stem cells, as describedherein.

FGFs are heparin-binding proteins, which interact withcell-surface-associated heparan sulfate proteoglycans to effect FGFsignaling. At least 22 different members of the FGF family have beenidentified. FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, andFGF10 bind and effect signaling through fibroblast growth receptors(FGFR).

FGFs induce mitosis in a variety of cell types and also have regulatory,morphological, and endocrine effects. FGFs function throughout embryonicdevelopment and aid in mesoderm induction, antero-posterior patterning,limb development, neural induction and neural development. In oneembodiment, a preferred FGF for use with the methods described herein isFGF7, which is also known in the art as Keratinocyte Growth Factor(KGF).

In some embodiments, the dosage range useful for FGF7 or FGF2 is between10 and 200 ng/mL, for example between 10 and 100 ng/mL, between 10 and50 ng/mL, between 15 and 200 ng/mL, between 20 and 200 ng/mL, between 30and 200 ng/mL, between 40 and 200 ng/mL, between 50 and 200 ng/mL,between 60 and 200 ng/mL, between 70 and 200 ng/mL, between 80 and 200ng/mL, between 90 and 200 ng/mL, between 100 and 200 ng/mL, or between150 and 200 ng/mL.

In some embodiments the dose of FGF7 or FGF2 is e.g., at least 10 ng/mL,at least 20 ng/mL, at least 30 ng/mL, at least 40 ng/mL, at least 50ng/mL, at least 60 ng/mL, at least 70 ng/mL, at least 80 ng/mL, at least90 ng/mL, at least 100 ng/mL, at least 110 ng/mL, at least 120 ng/mL, atleast 130 ng/mL, at least 140 ng/mL, at least 150 ng/mL, at least 160ng/mL, at least 170 ng/mL, at least 180 ng/mL, at least 190 ng/mL, atleast 200 ng/mL, at least 225 ng/mL, at least 250 ng/mL or more.

Wnt pathway modulation: Without wishing to be bound by theory, Wntproteins and their cognate receptors signal through at least twodistinct intracellular pathways. The “canonical” Wnt signaling pathway,(referred to herein as the Wnt/β-catenin pathway) involves Wnt signalingvia β-catenin to activate transcription through TCF-related proteins(van de Wetering et al. (2002) Cell 109 Suppl: S13-9; Moon et al. (2002)Science 296(5573): 1644-6). A non-canonical alternative pathway exists,in which Wnt activates protein kinase C (PKC),calcium/calmodulin-dependent kinase II (CaMKII), JNK and Rho-GTPases(Veeman et al. (2003) Dev Cell 5(3): 367-77), and is often involved inthe control of cell polarity.

Wnt Antagonists: Provided herein are methods for differentiating humanlung progenitor cells to a more differentiated stem cell phenotype,e.g., to an Nkx2.1+, Sox2+ proximal multipotent airway progenitor cellor to an Nkx2.1+, Sox9+ distal multipotent lung progenitor cell, or toan Nkx2.1+, p63+ multipotent airway basal stem cell by contacting a cellwith a Wnt antagonist.

As used herein, the term “Wnt antagonist” or “Wnt inhibitor” refers toany agent that inhibits the Wnt/β-catenin pathway, or enhances theactivity and/or expression of inhibitors of Wnt/β-catenin signaling, forexample activators or enhancers of GSK-3β activity. A Wnt inhibitoryagent as used herein can suppress the Wnt/β-catenin pathway at any pointalong the pathway, for example, but not limited to decreasing theexpression and/or activity of Wnt, or β-catenin or Wnt dependent genesand/or proteins, and increasing the expression and/or activity ofendogenous inhibitors of Wnt and/or β-catenin or increasing theexpression and/or activity of endogenous inhibitors of components of theWnt/β-catenin pathway, for example increasing the expression of GSK-3β.

Some non-limiting examples of Wnt antagonists include Wnt pathwayinhibitor V (also known as(E)-4-(2,6-Difluorostyryl)-N,N-dimethylaniline), IWR-1 endo, IWP-2,CCT036477, and a peptide comprising the sequencet-Boc-NH-Met-Asp-Gly-Cys-Glu-Leu-CO2H (SEQ ID NO: 1).

In some embodiments, the dosage range useful for a Wnt antagonist (e.g.IWR-1) is between 20 and 200 ng/mL, between 30 and 200 ng/mL, between 40and 200 ng/mL, between 50 and 200 ng/mL, between 60 and 200 ng/mL,between 70 and 200 ng/mL, between 80 and 200 ng/mL, between 90 and 200ng/mL, between 100 and 200 ng/mL, or between 150 and 200 ng/mL.

In some embodiments the dose of a Wnt antagonist is e.g., at least 20ng/mL, at least 30 ng/mL, at least 40 ng/mL, at least 50 ng/mL, at least60 ng/mL, at least 70 ng/mL, at least 80 ng/mL, at least 90 ng/mL, atleast 100 ng/mL, at least 110 ng/mL, at least 120 ng/mL, at least 130ng/mL, at least 140 ng/mL, at least 150 ng/mL, at least 160 ng/mL, atleast 170 ng/mL, at least 180 ng/mL, at least 190 ng/mL, at least 200ng/mL, or more.

Wnt agonists: Provided herein are methods for differentiating a humanforegut endoderm cell to a more differentiated cell type, e.g., to anNkx2.1+, Tuj negative, Pax8 negative lung progenitor cell by contactinga cell with a Wnt agonist.

As used herein, the term “Wnt agonist” refers to any agent thatactivates the Wnt/β-catenin pathway, or inhibits the activity and/orexpression of inhibitors of Wnt/β-catenin signaling, for exampleantagonists or inhibitors of GSK-3β activity. A Wnt activating agent asused herein can enhance signaling through the Wnt/β-catenin pathway atany point along the pathway, for example, but not limited to increasingthe expression and/or activity of Wnt, or β-catenin or Wnt dependentgenes and/or proteins, and decreasing the expression and/or activity ofendogenous inhibitors of Wnt and/or β-catenin or decreasing theexpression and/or activity of endogenous inhibitors of components of theWnt/β-catenin pathway, for example decreasing the expression of GSK-3β.

Some non-limiting examples of Wnt pathway agonists include CHIR9902,2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine,BIO, (2′Z,3′E)-6-Bromoindirubin-3′-oxime,5-(Furan-2-yl)-N-(3-(1H-imidazol-1-yl)propyl)-1,2-oxazole-3-carboxamide,and SKL2001.

In some embodiments, the dosage range useful for a Wnt agonist (e.g.CHIR9902) is between 20 and 200 ng/mL, between 30 and 200 ng/mL, between40 and 200 ng/mL, between 50 and 200 ng/mL, between 60 and 200 ng/mL,between 70 and 200 ng/mL, between 80 and 200 ng/mL, between 90 and 200ng/mL, between 100 and 200 ng/mL, or between 150 and 200 ng/mL.

In some embodiments the dose of a Wnt agonist is e.g., at least 20ng/mL, at least 30 ng/mL, at least 40 ng/mL, at least 50 ng/mL, at least60 ng/mL, at least 70 ng/mL, at least 80 ng/mL, at least 90 ng/mL, atleast 100 ng/mL, at least 110 ng/mL, at least 120 ng/mL, at least 130ng/mL, at least 140 ng/mL, at least 150 ng/mL, at least 160 ng/mL, atleast 170 ng/mL, at least 180 ng/mL, at least 190 ng/mL, at least 200ng/mL, or more.

In some embodiments, the dosage range useful for a Wnt agonist (e.g.,CHIR9902) is between 0.1 and 5 μM, for example, between 0.1 and 4 μM,between 0.1 and 3 μM, between 0.1 and 2 μM, between 0.1 and 1 μM,between 0.1 and 0.5 μM, between 0.5 and 3 μM, between 0.5 and 2 μM,between 0.5 and 1 μM, between 1 and 2 μM, between 1.5 and 2 μM, between1 and 1.5 μM, between 2 and 5 μM, between 3 and 5 μM, between 4 and 5μM.

In some embodiments, the dose of a Wnt agonist (e.g., CHIR9902) is e.g.,at least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 1.1 μM, atleast 1.2 μM, at least 1.3 μM, at least 1.4 μM, at least 1.5 μM, atleast 1.6 μM, at least 1.7 μM, at least 1.8 μM, at least 1.9 μM, atleast 2 μM, at least 2.5 μM, at least 3 μM, at least 4 μM, at least 5 μMor more.

PI3 Kinase Inhibitors:

Phosphoinositide 3-kinases (PI3K) are lipid kinases that phosphorylatelipids at the 3-hydroxyl residue of an inositol ring (Whitman et al(1988) Nature, 332:664). The 3-phosphorylated phospholipids (PIP3s)generated by PI3-kinases act as second messengers recruiting kinaseswith lipid binding domains (including plekstrin homology (PH) regions),such as Akt and phosphoinositide-dependent kinase-1 (PDK1). Binding ofAkt to membrane PIP3s causes the translocation of Akt to the plasmamembrane, bringing Akt into contact with PDK1, which is responsible foractivating Akt. The tumor-suppressor phosphatase, PTEN, dephosphorylatesPIP3 and therefore acts as a negative regulator of Akt activation. ThePI3-kinases Akt and PDK1 are important in the regulation of manycellular processes including cell cycle regulation, proliferation,survival, apoptosis and motility and are significant components of themolecular mechanisms of diseases such as cancer, diabetes and immuneinflammation (Vivanco et al (2002) Nature Rev. Cancer 2:489; Phillips etal (1998) Cancer 83:41).

As used herein, the term “PI3 kinase inhibitor” or “PI3 kinaseantagonist” refers to any agent that inhibits the activity of PI3kinase. Some non-limiting examples of a PI3 kinase inhibitor useful withthe methods described herein include LY294002, wortmannin, PIK-75,ZSTK474, and Pp242.

In some embodiments, the dosage range useful for a PI3 kinase inhibitor(e.g., ZSTK474, or PIK-75) is between 0.1 and 5 μM, for example, between0.1 and 4 μM, between 0.1 and 3 μM, between 0.1 and 2 μM, between 0.1and 1 μM, between 0.1 and 0.5 μM, between 0.5 and 3 μM, between 0.5 and2 μM, between 0.5 and 1 μM, between 1 and 2 μM, between 1.5 and 2 μM,between 1 and 1.5 μM, between 2 and 5 μM, between 3 and 5 μM, between 4and 5 μM.

In some embodiments, the dose of a PI3 kinase inhibitor (e.g., ZSTK474,or PIK-75) is e.g., at least 0.1 μM, at least 0.5 μM, at least 1 μM, atleast 1.1 μM, at least 1.2 μM, at least 1.3 μM, at least 1.4 μM, atleast 1.5 μM, at least 1.6 μM, at least 1.7 μM, at least 1.8 μM, atleast 1.9 μM, at least 2 μM, at least 2.5 μM, at least 3 μM, at least 4μM, at least 5 μM or more.

Monitoring Differentiation of Human Lung Progenitors

Provided herein are methods for differentiating or redifferentiating apluripotent stem cell (e.g., an anterior foregut endoderm cell, adefinitive endoderm cell, an ES cell or an iPSC) to a human lungprogenitor cell, and optionally further differentiating such human lungprogenitor cells to lung airway cells, such as basal cells, Clara cells,ciliated cells and/or goblet cells. These aspects are based on the noveldiscovery of a method for differentiation of pluripotent stem cells to astem cell or progenitor cell committed to the lung and/or airwaylineage. Such methods are exemplified in the Examples section herein.Also provided herein are compositions of human lung progenitor cellshaving particular characteristics, such as the presence of one or morecell surface or other markers that are lung cell specific.Alternatively, or in addition, the human lung progenitor cellcompositions described herein lack markers of embryonic stem cells orinduced pluripotent stem cells. In one embodiment of the methodsdescribed herein, one or more cell surface markers are used to determinethe degree of differentiation along the spectrum of embryonic stem cellsor iPSCs to fully differentiated lung cells.

Cell surface markers, particularly stem cell surface markers, are usefulwith the methods and compositions described herein to identify thedifferentiation or dedifferentiation state of a cell. For example,during reprogramming of a somatic cell to an induced pluripotent stemcell the activation of stem cell markers can be used to confirm that thesomatic cell has been dedifferentiated (either partially or completely).Alternatively, during differentiation of an ES cell or an iPSC to ahuman lung progenitor cell, the activation of lung-specific markers canbe used to confirm the degree of differentiation that the stem cell hasundergone. In addition, the activation or deactivation of particularlung-specific markers can be used to determine the degree ofmultipotency of a human lung progenitor cell. This can be achieved bycomparing the lung-specific markers present on, or expressed by the cellwith the marker profile of lung cells during development and inferringthe degree of multipotency of the differentiated cell based on the knowndegree of multipotency of the corresponding lung cell during embryonicdevelopment.

Marker-specific agents can be used to recognize stem cell markers, forinstance labeled antibodies that recognize and bind to cell-surfacemarkers or antigens on desired stem cells. Antibodies or similar agentsspecific for a given marker, or set of markers, can be used to separateand isolate the desired stem cells using fluorescent activated cellsorting (FACS), panning methods, magnetic particle selection, particlesorter selection and other methods known to persons skilled in the art,including density separation (Xu et al. (2002) Circ. Res. 91:501;U.S.S.N. 20030022367) and separation based on other physical properties(Doevendans et al. (2000) J. Mol. Cell. Cardiol. 32:839-851).

Alternatively, genetic selection methods can be used, where a progenitoror stem cell can be genetically engineered to express a reporter proteinoperatively linked to a tissue-specific promoter and/or a specific genepromoter; therefore the expression of the reporter can be used forpositive selection methods to isolate and enrich the desired stem cell.For example, a fluorescent reporter protein can be expressed in thedesired stem cell by genetic engineering methods to operatively link themarker protein to a promoter active in a desired stem cell (Klug et al.(1996) J. Clin. Invest. 98:216-224; U.S. Pat. No. 6,737,054). In someembodiments, cells from which the human lung progenitor cells arederived are not modified using genetic means. Other approaches forpositive selection include drug selection, for instance as described byKlug et al., supra, involving enrichment of desired cells by densitygradient centrifugation. Negative selection can be performed, selectingand removing cells with undesired markers or characteristics, forexample fibroblast markers, epithelial cell markers etc.

Undifferentiated ES cells express genes that can be used as markers todetect the presence of undifferentiated cells. The polypeptide productsof such genes can be used as markers for negative selection. Forexample, see U.S.S.N. 2003/0224411 A1; Bhattacharya (2004) Blood103(8):2956-64; and Thomson (1998), supra., each herein incorporated byreference. Human ES cell lines express cell surface markers thatcharacterize undifferentiated nonhuman primate ES and human EC cells,including, but not limited to, stage-specific embryonic antigen(SSEA)-3, SSEA-4, TRA-I-60, TRA-1-81, and alkaline phosphatase. Theglobo-series glycolipid GL7, which carries the SSEA-4 epitope, is formedby the addition of sialic acid to the globo-series glycolipid Gb5, whichcarries the SSEA-3 epitope. Thus, GL7 reacts with antibodies to bothSSEA-3 and SSEA-4. Undifferentiated human ES cell lines do not stain forSSEA-1, but differentiated cells stain strongly for SSEA-1. Methods forproliferating hES cells in the undifferentiated form are described in WO99/20741, WO 01/51616, and WO 03/020920, the contents of which areherein incorporated by reference in their entireties.

Exemplary cell surface markers expressed on lung progenitor cellsinclude, but are not limited to, Sox2, Sox9, p63, FoxP2, ETV4/5, FoxA2,Nkx2.1, Gata6, ID2, CK5, NGFR, FoxJ1, CCSP, Scgb3a2, Muc5ac, T1a, Spc,and Scgn. Particular cell compositions with combinations of such cellsurface markers are exemplified herein in the Examples section.

In some embodiments, the human lung progenitor cells are an enrichedpopulation of cells; that is, the percentage of human lung progenitorcells (e.g., percent of cells) in a population of cells is at least 10%of the total number of cells in the population. For example, an enrichedpopulation comprises at least 15% human lung progenitor cells, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99% or even 100%of the population comprises human lung progenitor cells. In someembodiments, a population of cells comprises at least 100 cells, atleast 500 cells, at least 1000 cells, at least 1×10⁴ cells, at least1×10⁵ cells, at least 1×10⁶ cells, at least 1×10⁷ cells, at least 1×10⁸cells, at least 1×10⁹ cells, at least 1×10¹⁰ cells, at least 1×10¹¹cells, at least 1×10¹² cells, at least 1×10¹³ cells, at least 1×10¹⁴cells, at least 1×10¹⁵ cells, or more.

In one embodiment, the human lung progenitor cells described herein arenot tumor cells or cancer cells. In such embodiments, the human lungprogenitor cell can be distinguished from a tumor cell or cancer cellusing e.g., a cell marker profile.

Scaffold Compositions

Biocompatible synthetic, natural, as well as semi-synthetic polymers,can be used for synthesizing polymeric particles that can be used as ascaffold material. In general, for the practice of the methods describedherein, it is preferable that a scaffold biodegrades such that the lungprogenitor cells can be isolated from the polymer prior to implantationor such that the scaffold degrades over time in a subject and does notrequire removal. Thus, in one embodiment, the scaffold provides atemporary structure for growth and/or delivery of human lung progenitorcells to a subject in need thereof. In some embodiments, the scaffoldpermits human cell progenitors to be grown in a shape suitable fortransplantation or administration into a subject in need thereof,thereby permitting removal of the scaffold prior to implantation andreducing the risk of rejection or allergic response initiated by thescaffold itself.

Examples of polymers which can be used include natural and syntheticpolymers, although synthetic polymers are preferred for reproducibilityand controlled release kinetics. Synthetic polymers that can be usedinclude biodegradable polymers such as poly(lactide) (PLA),poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), and otherpolyhydroxyacids, poly(caprolactone), polycarbonates, polyamides,polyanhydrides, polyphosphazene, polyamino acids, polyortho esters,polyacetals, polycyanoacrylates and biodegradable polyurethanes;non-biodegradable polymers such as polyacrylates, ethylene-vinyl acetatepolymers and other acyl-substituted cellulose acetates and derivativesthereof; polyurethanes, polystyrenes, polyvinyl chloride, polyvinylfluoride, poly(vinyl imidazole), chlorosulphonated polyolefins, andpolyethylene oxide. Examples of biodegradable natural polymers includeproteins such as albumin, collagen, fibrin, silk, synthetic polyaminoacids and prolamines; polysaccharides such as alginate, heparin; andother naturally occurring biodegradable polymers of sugar units.Alternately, combinations of the aforementioned polymers can be used.

PLA, PGA and PLA/PGA copolymers are particularly useful for formingbiodegradable scaffolds. PLA polymers are usually prepared from thecyclic esters of lactic acids. Both L(+) and D(−) forms of lactic acidcan be used to prepare the PLA polymers, as well as the opticallyinactive DL-lactic acid mixture of D(−) and L(+) lactic acids. Methodsof preparing polylactides are well documented in the patent literature.The following U.S. Patents, the teachings of which are herebyincorporated by reference, describe in detail suitable polylactides,their properties and their preparation: U.S. Pat. No. 1,995,970 toDorough; U.S. Pat. No. 2,703,316 to Schneider; U.S. Pat. No. 2,758,987to Salzberg; U.S. Pat. No. 2,951,828 to Zeile; U.S. Pat. No. 2,676,945to Higgins; and U.S. Pat. Nos. 2,683,136; 3,531,561 to Trehu.

PGA is a homopolymer of glycolic acid (hydroxyacetic acid). In theconversion of glycolic acid to poly(glycolic acid), glycolic acid isinitially reacted with itself to form the cyclic ester glycolide, whichin the presence of heat and a catalyst is converted to a high molecularweight linear-chain polymer. PGA polymers and their properties aredescribed in more detail in Cyanamid Research Develops World's FirstSynthetic Absorbable Suture”, Chemistry and Industry, 905 (1970).

Fibers can be formed by melt-spinning, extrusion, casting, or othertechniques well known in the polymer processing area. Preferredsolvents, if used to remove a scaffold prior to implantation, are thosewhich are completely removed by the processing or which arebiocompatible in the amounts remaining after processing.

Polymers for use in the matrix should meet the mechanical andbiochemical parameters necessary to provide adequate support for thecells with subsequent growth and proliferation. The polymers can becharacterized with respect to mechanical properties such as tensilestrength using an Instron tester, for polymer molecular weight by gelpermeation chromatography (GPC), glass transition temperature bydifferential scanning calorimetry (DSC) and bond structure by infrared(IR) spectroscopy.

Scaffolds can be of any desired shape and can comprise a wide range ofgeometries that are useful for the methods described herein. Anon-limiting list of shapes includes, for example, hollow particles,tubes, sheets, cylinders, spheres, and fibers, among others. The shapeor size of the scaffold should not substantially impede cell growth,cell differentiation, cell proliferation or any other cellular process,nor should the scaffold induce cell death via e.g., apoptosis ornecrosis. In addition, care should be taken to ensure that the scaffoldshape permits appropriate surface area for delivery of nutrients fromthe surrounding medium to cells in the population, such that cellviability is not impaired. The scaffold porosity can also be varied asdesired by one of skill in the art.

In some embodiments, attachment of the cells to a polymer is enhanced bycoating the polymers with compounds such as basement membranecomponents, agar, agarose, gelatin, gum arabic, collagens types I, II,III, IV, and V, fibronectin, laminin, glycosaminoglycans, polyvinylalcohol, mixtures thereof, and other hydrophilic and peptide attachmentmaterials known to those skilled in the art of cell culture or tissueengineering. Examples of a material for coating a polymeric scaffoldinclude polyvinyl alcohol and collagen.

In some embodiments, the scaffold can include decellularized lungtissue. Methods for producing decellularized lung tissue are known inthe art, see e.g., WO2011/005306. Briefly, the process ofdecellularization involves chemically stripping lung tissue of its cellsand removing the cellular debris, which leaves behind the structure ofthe extracellular matrix. The extracellular matrix can then berepopulated with human lung progenitor cells as described herein, andoptionally with other bioactive agents. Such decellularized scaffoldscan be prepared from a portion of the subject's own lung and thereforethe risk of rejection or allergic reaction in response to therepopulated and administered scaffold can be minimized.

In some embodiments it can be desirable to add bioactive molecules tothe scaffold. A variety of bioactive molecules can be delivered usingthe matrices described herein. These are referred to generically hereinas “factors” or “bioactive factors”.

In one embodiment, the bioactive factors include growth factors.Examples of growth factors include platelet derived growth factor(PDGF), transforming growth factor alpha or beta (TGFβ), bonemorphogenic protein 4 (BMP4), fibroblastic growth factor 7 (FGF7),fibroblast growth factor 10 (FGF10), epidermal growth factor (EGF/TGFα),vascular endothelium growth factor (VEGF), some of which are alsoangiogenic factors.

These factors are known to those skilled in the art and are availablecommercially or described in the literature. Bioactive molecules can beincorporated into the matrix and released over time by diffusion and/ordegradation of the matrix, or they can be suspended with the cellsuspension.

Treatment of Lung Disease/Disorders and Lung Injury

The methods and compositions provided herein relate to the generationand use of human lung progenitor cells. Accordingly, provided herein aremethods for the treatment and prevention of a lung injury or a lungdisease or disorder in a subject in need thereof. The methods describedherein can be used to treat, ameliorate, prevent or slow the progressionof a number of lung diseases or their symptoms, such as those resultingin pathological damage to lung or airway architecture and/or alveolardamage. The terms “respiratory disorder,” “respiratory disease,” “lungdisease,” “lung disorder,” “pulmonary disease,” and “pulmonarydisorder,” are used interchangeably herein and refer to any conditionand/or disorder relating to respiration and/or the respiratory system,including the lungs, pleural cavity, bronchial tubes, trachea, upperrespiratory tract, airways, or other components or structures of theairway system.

Such lung diseases include, but are not limited to, bronchopulmonarydysplasia (BPD), chronic obstructive pulmonary disease (COPD), cysticfibrosis, bronchiectasis, cor pulmonale, pneumonia, lung abcess, acutebronchitis, chronic bronchitis, emphysema, pneumonitis (e.g.,hypersensitivity pneumonitis or pneumonitis associated with radiationexposure), alveolar lung diseases and interstitial lung diseases,environmental lung disease (e.g., associated with asbestos, fumes or gasexposure), aspiration pneumonia, pulmonary hemorrhage syndromes,amyloidosis, connective tissue diseases, systemic sclerosis, ankylosingspondylitis, pulmonary actinomycosis, pulmonary alveolar proteinosis,pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonaryinflammation, pulmonary histiocytosis X, pulmonary hypertension,surfactant deficiencies, pulmonary hypoplasia, pulmonary neoplasia,pulmonary nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusivedisease, rheumatoid lung disease, sarcoidosis, post-pneumonectomy,Wegener's granulomatosis, allergic granulomatosis, granulomatousvasculitides, eosinophilia, asthma and airway hyperreactivity (AHR)(e.g., mild intermittent asthma, mild persistent asthma, moderatepersistent asthma, severe persistent asthma, acute asthma, chronicasthma, atopic asthma, allergic asthma or idiosyncratic asthma),allergic bronchopulmonary aspergillosis, chronic sinusitis, pancreaticinsufficiency, lung or vascular inflammation, bacterial or viralinfection, e.g., Haemophilus influenzae, S. aureus, Pseudomonasaeruginosa or respiratory syncytial virus (RSV) infection or an acute orchronic adult or pediatric respiratory distress syndrome (RDS) such asgrade I, II, III or IV RDS or an RDS associated with, e.g., sepsis,pneumonia, reperfusion, atelectasis or chest trauma.

Chronic obstructive pulmonary diseases (COPDs) include those conditionswhere airflow obstruction is located at upper airways,intermediate-sized airways, bronchioles or parenchyma, which can bemanifested as, or associated with, tracheal stenosis, tracheal rightventricular hypertrophy pulmonary hypertension, polychondritis,bronchiectasis, bronchiolitis, e.g., idiopathic bronchiolitis, ciliarydyskinesia, asthma, emphysema, connective tissue disease, bronchiolitisof chronic bronchitis or lung transplantation.

The methods described herein can also be used to treat or ameliorateacute or chronic lung diseases/disorders or their symptoms orcomplications, including airway epithelium injury, airway smooth musclespasm or airway hyperresponsiveness, airway mucosa edema, increasedmucus secretion, excessive T cell activation, or desquamation,atelectasis, cor pulmonale, pneumothorax, subcutaneous emphysema,dyspnea, coughing, wheezing, shortness of breath, tachypnea, fatigue,decreased forced expiratory volume in the 1st second (FEV₁), arterialhypoxemia, respiratory acidosis, inflammation including unwantedelevated levels of mediators such as IL-4, IL-5, IgE, histamine,substance P, neurokinin A, calcitonin gene-related peptide orarachidonic acid metabolites such as thromboxane or leukotrienes (LTD₄or LTC₄), and cellular airway wall infiltration, e.g., by eosinophils,lymphocytes, macrophages or granulocytes.

Any of these and other respiratory or pulmonary conditions or symptomsare known in the art. See e.g., The Merck Manual, 17th edition, M. H.Beers and R. Berkow editors, 1999, Merck Research Laboratories,Whitehouse Station, N.J., ISBN 0911910-10-7, or in other referencescited herein.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably in the context of the placementof cells, e.g. lung progenitor cells, as described herein into asubject, by a method or route which results in at least partiallocalization of the introduced cells at a desired site, such as a siteof injury or repair, such that a desired effect(s) is produced. Thecells e.g. lung progenitor cells, or their differentiated progeny (e.g.airway progenitor cells, basal cells, Clara cells, ciliated cells orgoblet cells) can be implanted directly to the respiratory airways, oralternatively be administered by any appropriate route which results indelivery to a desired location in the subject where at least a portionof the implanted cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years, i.e., long-term engraftment. For example, insome embodiments of the aspects described herein, an effective amount oflung progenitor cells is administered directly to the lungs of an infantsuffering from bronchopulmonary dysplasia by intratrachealadministration. In other embodiments, lung progenitor cells can beadministered via an indirect systemic route of administration, such asan intraperitoneal or intravenous route.

When provided prophylactically, lung progenitor cells described hereincan be administered to a subject in advance of any symptom of a lungdisorder, e.g., an asthma attack or to a premature infant. Accordingly,the prophylactic administration of a lung progenitor cell populationserves to prevent a lung disorder, as disclosed herein.

When provided therapeutically, lung progenitor cells are provided at (orafter) the onset of a symptom or indication of a lung disorder, e.g.,upon the onset of COPD.

In some embodiments of the aspects described herein, the lung progenitorcell population being administered according to the methods describedherein comprises allogeneic lung progenitor cells obtained from one ormore donors. As used herein, “allogeneic” refers to a lung progenitorcell or biological samples comprising lung progenitor cells obtainedfrom one or more different donors of the same species, where the genesat one or more loci are not identical. For example, a lung progenitorcell population being administered to a subject can be derived fromumbilical cord blood obtained from one more unrelated donor subjects, orfrom one or more non-identical siblings. In some embodiments, syngeneiclung progenitor cell populations can be used, such as those obtainedfrom genetically identical animals, or from identical twins. In otherembodiments of this aspect, the lung progenitor cells are autologouscells; that is, the lung progenitor cells are obtained or isolated froma subject and administered to the same subject, i.e., the donor andrecipient are the same.

Depending on the disease/disorder or injury to be treated, as well asthe location of the lung injury, either an undifferentiated human lungprogenitor cell, or a differentiated cell thereof can be administered tothe subject.

Pharmaceutically Acceptable Carriers

The methods of administering human lung progenitors to a subject asdescribed herein involve the use of therapeutic compositions comprisinglung progenitor cells. Therapeutic compositions contain aphysiologically tolerable carrier together with the cell composition andoptionally at least one additional bioactive agent as described herein,dissolved or dispersed therein as an active ingredient. In a preferredembodiment, the therapeutic composition is not substantially immunogenicwhen administered to a mammal or human patient for therapeutic purposes,unless so desired. As used herein, the terms “pharmaceuticallyacceptable”, “physiologically tolerable” and grammatical variationsthereof, as they refer to compositions, carriers, diluents and reagents,are used interchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset,transplant rejection, allergic reaction, and the like. Apharmaceutically acceptable carrier will not promote the raising of animmune response to an agent with which it is admixed, unless so desired.The preparation of a composition that contains active ingredientsdissolved or dispersed therein is well understood in the art and neednot be limited based on formulation. Typically such compositions areprepared as injectable either as liquid solutions or suspensions,however, solid forms suitable for solution, or suspensions, in liquidprior to use can also be prepared.

In general, the human lung progenitor cells described herein areadministered as a suspension with a pharmaceutically acceptable carrier.One of skill in the art will recognize that a pharmaceuticallyacceptable carrier to be used in a cell composition will not includebuffers, compounds, cryopreservation agents, preservatives, or otheragents in amounts that substantially interfere with the viability of thecells to be delivered to the subject. A formulation comprising cells caninclude e.g., osmotic buffers that permit cell membrane integrity to bemaintained, and optionally, nutrients to maintain cell viability orenhance engraftment upon administration. Such formulations andsuspensions are known to those of skill in the art and/or can be adaptedfor use with the human lung progenitor cells as described herein usingroutine experimentation.

A cell composition can also be emulsified or presented as a liposomecomposition, provided that the emulsification procedure does notadversely affect cell viability. The cells and any other activeingredient can be mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient and in amountssuitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition as described herein caninclude pharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions as described herein that is effective in the treatment of aparticular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques.

Administration and Efficacy

Provided herein are methods for treating a lung disease, a lungdisorder, or a lung injury comprising administering human lungprogenitor cells or differentiated progeny thereof to a subject in needthereof.

Measured or measurable parameters include clinically detectable markersof disease, for example, elevated or depressed levels of a clinical orbiological marker, as well as parameters related to a clinicallyaccepted scale of symptoms or markers for a disease or disorder. It willbe understood, however, that the total daily usage of the compositionsand formulations as disclosed herein will be decided by the attendingphysician within the scope of sound medical judgment. The exact amountrequired will vary depending on factors such as the type of diseasebeing treated.

The term “effective amount” as used herein refers to the amount of apopulation of human lung progenitor cells or their progeny needed toalleviate at least one or more symptom of the lung injury or the lungdisease or disorder, and relates to a sufficient amount of a compositionto provide the desired effect, e.g., treat a subject having smokinginduced-injury or cystic fibrosis. The term “therapeutically effectiveamount” therefore refers to an amount of human lung progenitor cells ora composition comprising human lung progenitor cells that is sufficientto promote a particular effect when administered to a typical subject,such as one who has or is at risk for a lung disease or disorder. Aneffective amount as used herein would also include an amount sufficientto prevent or delay the development of a symptom of the disease, alterthe course of a symptom disease (for example but not limited to, slowthe progression of a symptom of the disease), or reverse a symptom ofthe disease. It is understood that for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the artusing routine experimentation.

In some embodiments, the subject is first diagnosed as having a diseaseor disorder affecting the lung tissue prior to administering the cellsaccording to the methods described herein. In some embodiments, thesubject is first diagnosed as being at risk of developing lung diseaseor disorder prior to administering the cells. For example, a prematureinfant can be at a significant risk of developing a lung disease ordisorder.

For use in the various aspects described herein, an effective amount ofhuman lung progenitor cells, comprises at least 10² lung progenitorcells, at least 5×10² lung progenitor cells, at least 10³ lungprogenitor cells, at least 5×10³ lung progenitor cells, at least 10⁴lung progenitor cells, at least 5×10⁴ lung progenitor cells, at least10⁵ lung progenitor cells, at least 2×10⁵ lung progenitor cells, atleast 3×10⁵ lung progenitor cells, at least 4×10⁵ lung progenitor cells,at least 5×10⁵ lung progenitor cells, at least 6×10⁵ lung progenitorcells, at least 7×10⁵ lung progenitor cells, at least 8×10⁵ lungprogenitor cells, at least 9×10⁵ lung progenitor cells, at least 1×10⁶lung progenitor cells, at least 2×10⁶ lung progenitor cells, at least3×10⁶ lung progenitor cells, at least 4×10⁶ lung progenitor cells, atleast 5×10⁶ lung progenitor cells, at least 6×10⁶ lung progenitor cells,at least 7×10⁶ lung progenitor cells, at least 8×10⁶ lung progenitorcells, at least 9×10⁶ lung progenitor cells, or multiples thereof. Thelung progenitor cells can be derived from one or more donors, or can beobtained from an autologous source. In some embodiments of the aspectsdescribed herein, the lung progenitor cells are expanded in cultureprior to administration to a subject in need thereof.

Exemplary modes of administration for use in the methods describedherein include, but are not limited to, injection, intrapulmonary(including intranasal and intratracheal) infusion, inhalation as anaerosol (including intranasal), and implantation (with or without ascaffold material). “Injection” includes, without limitation,intravenous, intramuscular, intraarterial, intradermal, intraperitoneal,transtracheal and subcutaneous. The phrases “parenteral administration”and “administered parenterally” as used herein, refer to modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous,intraperitoneal, intramuscular, intraarterial, intradermal,transtracheal, and subcutaneous administration.

In some embodiments, a therapeutically effective amount of lungprogenitor cells is administered using intrapulmonary administration,such as an intranasal or intratracheal route. In some aspects of thesemethods, a therapeutically effective amount of lung progenitor cells areadministered using a systemic, such as an intraperitoneal or intravenousroute. In other aspects of these methods, a therapeutically effectiveamount of lung progenitor cells is administered using bothintrapulmonary and intraperitoneal administration. These methods areparticularly aimed at therapeutic and prophylactic treatments of humansubjects having, or at risk of having, a lung disease or disorder. Thehuman lung progenitor cells described herein can be administered to asubject having any lung disease or disorder by any appropriate routewhich results in an effective treatment in the subject. In someembodiments of the aspects described herein, a subject having a lungdisorder is first selected prior to administration of the cells.

In some embodiments, an effective amount of lung progenitor cells areadministered to a subject by intrapulmonary administration or delivery.As defined herein, “intrapulmonary” administration or delivery refers toall routes of administration whereby a population of lung progenitorcells, such as Nkx2.1+, Sox2+ lung progenitor cells, is administered ina way that results in direct contact of these cells with the airways ofa subject, including, but not limited to, transtracheal, intratracheal,and intranasal administration. In some such embodiments, the cells areinjected into the nasal passages or trachea. In some embodiments, thecells are directly inhaled by a subject. In some embodiments,intrapulmonary delivery of cells includes administration methods wherebycells are administered, for example as a cell suspension, to anintubated subject via a tube placed in the trachea or “trachealintubation.”

As used herein, “tracheal intubation” refers to the placement of aflexible tube, such as a plastic tube, into the trachea. The most commontracheal intubation, termed herein as “orotracheal intubation” is where,with the assistance of a laryngoscope, an endotracheal tube is passedthrough the mouth, larynx, and vocal cords, into the trachea. A bulb isthen inflated near the distal tip of the tube to help secure it in placeand protect the airway from blood, vomit, and secretions. In someembodiments, cells are administered to a subject having “nasotrachealintubation,” which is defined as a tracheal intubation where a tube ispassed through the nose, larynx, vocal cords, and trachea.

In some embodiments, an effective amount of lung progenitor cells isadministered to a subject by systemic administration, such asintravenous administration.

The phrases “systemic administration,” “administered systemically”,“peripheral administration” and “administered peripherally” as usedherein refer to the administration of a population of lung progenitorcells other than directly into a target site, tissue, or organ, such asthe lung, such that it enters, instead, the subject's circulatory systemand, thus, is subject to metabolism and other like processes.

In some embodiments of the aspects described herein, one or more routesof administration are used in a subject to achieve distinct effects. Forexample, lung progenitor cells can be administered to a subject by bothintratracheal and intraperitoneal administration routes for treating orrepairing lung epithelium and for pulmonary vascular repair andregeneration respectively. In such embodiments, different effectiveamounts of the isolated or enriched lung progenitor cells can be usedfor each administration route.

Where aerosol administration is to be used, nebulizer devices requireformulations suitable for dispensing the particular composition. Thechoice of formulation will depend upon the specific composition used andthe number of lung progenitors to be administered; such formulations canbe adjusted by the skilled practitioner. However, as an example, wherethe composition is lung progenitor cells in a pharmaceuticallyacceptable carrier, the composition can be a suspension of the cells inan appropriate buffer (e.g., saline buffer) at an effectiveconcentration of cells per mL of solution. The formulation can alsoinclude cell nutrients, a simple sugar (e.g., for osmotic pressureregulation) or other components to maintain the viability of the cells.

Typically, each formulation for aerosol delivery via a nebulizer isspecific to the type of device employed and can involve the use of anappropriate propellant material, in addition to the usual diluents,adjuvants and/or carriers useful in therapy.

In some embodiments, additional agents to aid in treatment of thesubject can be administered before or following treatment with the lungprogenitor cells described herein. Such additional agents can be used toprepare the lung tissue for administration of the progenitor cells.Alternatively the additional agents can be administered after the lungprogenitor cells to support the engraftment and growth of theadministered cell in the damaged lung. Such additional agents can beformulated for use with a metered-dose inhaler device, which generallycomprises a finely divided powder containing a protein or small moleculesuspended in a propellant with the aid of a surfactant. The propellantcan be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid can also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device can comprise afinely divided dry powder containing proteins or small molecules and canalso include a bulking agent, such as lactose, sorbitol, sucrose, ormannitol in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation. Protein agentsshould most advantageously be prepared in particulate form with anaverage particle size of less than 10 μm (or microns), most preferably0.5 to 5 μm, for most effective delivery to the distal lung.

Nasal delivery of protein or other agents in addition to the lungprogenitor cells or progeny thereof is also contemplated. Nasal deliveryallows the passage of the protein or other agent to the blood streamdirectly after administering the therapeutic product to the nose,without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

The efficacy of treatment can be determined by the skilled clinician.However, a treatment is considered “effective treatment,” as the term isused herein, if any one or all of the symptoms, or other clinicallyaccepted symptoms or markers of lung disease, lung injury and/or a lungdisorder are reduced, e.g., by at least 10% following treatment with acomposition comprising human lung progenitor cells as described herein.Methods of measuring these indicators are known to those of skill in theart and/or described herein.

Indicators of lung disease or lung disorder, or lung injury includefunctional indicators, e.g., measurement of lung capacity and function,and oxygen saturation (e.g., tissue oxygen saturation or systemicarterial oxygen saturation), as well as biochemical indicators.

For idiopathic pulmonary fibrosis, for example, improved symptomsinclude an increase of at least 10% of predicted forced vital capacity(FVC) relative to values prior to treatment. FVC is the total volume ofair expired after a full inspiration. Patients with obstructive lungdisease usually have a normal or only slightly decreased vital capacity.Patients with restrictive lung disease have a decreased vital capacity.

Another measure is FEV1 (Forced Expiratory Volume in 1 Second). This isthe volume of air expired in the first second during maximal expiratoryeffort. The FEV1 is reduced in both obstructive and restrictive lungdisease. The FEV1 is reduced in obstructive lung disease because ofincreased airway resistance. It is reduced in restrictive lung diseasebecause of the low vital capacity.

A related measure is FEV1/FVC. This is the percentage of the vitalcapacity which is expired in the first second of maximal expiration. Inhealthy patients the FEV1/FVC is usually around 70%. In patients withobstructive lung disease FEV1/FVC decreases and can be as low as 20-30%in severe obstructive airway disease. Restrictive disorders have a nearnormal FEV1/FVC.

Where necessary or desired, animal models of lung injury or lung diseasecan be used to gauge the effectiveness of a particular composition asdescribed herein. As one example, the bleomycin-induced lung injurymodel of acute lung injury (ALI) can be used. Animal models of lungfunction are useful for monitoring bronchoconstriction, allergicresponse, late airway hyperresponsiveness in response to inhaledallergens, among other endpoints and can include, for example, head-outplethysmography or body-plethysmography models (see e.g., Hoymann, H Get al., J Pharmacol Toxicol Methods (2007) 55(1):16-26). Exemplaryanimal models for asthma, including models of allergic asthma (e.g.,acute and chronic allergic asthma), are known in the art. See e.g.,Nials and Uddin. (2008) Dis Model Mech 1:213-220; Zosky and Sly (2007)Clin Exp Allergy 37(7):973-88; and Kumar and Foster. (2002) Am J RespirCell Mol Biol 27(3):267-72. Animal models of pneumonia are reviewed byMizgerd and Skerrett (2008) Am J Physiol Lung Cell Mol Physiol294:L387-L398. In addition, small animal imaging can be applied to lungpathophysiologies (Brown R H, et al., Proc Am Thorac Soc (2008)5:591-600).

Screening Assays

The compositions described herein are useful to screen for agents forinducing differentiation of human lung progenitor cells or for thetreatment of a lung disease or disorder.

In some embodiments, the isolated human lung progenitor cells orisolated human disease-specific lung cells derived from such human lungprogenitor cells can be used in methods, assays, systems and kits todevelop specific in vitro assays. Such assays for drug screening andtoxicology studies have an advantage over existing assays because theyare of human origin, and do not require immortalization of cell lines,nor do they require tissue from cadavers, which poorly reflect thephysiology of normal human cells. For example, the methods, assays,systems, and kits described herein can be used to identify and/or testagents that can promote differentiation along the lung lineage. Inaddition to, or alternatively, the methods, assays, systems, and kitscan be used to identify and/or test for agents useful in treating a lungdisease or disorder, or for preventing/treating a lung injury.

Accordingly, provided herein are methods for screening a test compoundfor biological activity, the method comprising (a) contacting anisolated human lung progenitor cell as described herein, or its progeny,with a test compound and (b) determining any effect of the compound onthe cell. In one embodiment, the screening method further comprisesgenerating a human lung progenitor cell or a human lung disease-specificcell as disclosed herein. In one embodiment, the lung progenitor cell isfirst differentiated to a desired lung cell phenotype. The effect on thecell can be one that is observable directly or indirectly by use ofreporter molecules.

As used herein, the term “biological activity” or “bioactivity” refersto the ability of a test compound to affect a biological sample.Biological activity can include, without limitation, elicitation of astimulatory, inhibitory, regulatory, toxic or lethal response in abiological assay. For example, a biological activity can refer to theability of a compound to modulate the effect of an enzyme, block areceptor, stimulate a receptor, modulate the expression level of one ormore genes, modulate cell proliferation, modulate cell division,modulate cell metabolism, modulate differentiation, modulate cellmorphology, or a combination thereof. In some instances, a biologicalactivity can refer to the ability of a test compound to produce a toxiceffect in a biological sample.

As discussed above, the specific lineage can be a lineage which isphenotypic and/or genotypic of a disease (e.g., a lung disease).Alternatively, the specific lineage can be a lineage which is phenotypicand/or genotypic of an organ and/or tissue or a part thereof (e.g.,lung).

As used herein, the term “test compound” or “candidate agent” refers toan agent or collection of agents (e.g., compounds) that are to bescreened for their ability to have an effect on the cell. Test compoundscan include a wide variety of different compounds, including chemicalcompounds, mixtures of chemical compounds, e.g., polysaccharides, smallorganic or inorganic molecules (e.g. molecules having a molecular weightless than 2000 Daltons, less than 1000 Daltons, less than 1500 Dalton,less than 1000 Daltons, or less than 500 Daltons), biologicalmacromolecules, e.g., peptides, proteins, peptide analogs, and analogsand derivatives thereof, peptidomimetics, nucleic acids, nucleic acidanalogs and derivatives, an extract made from biological materials suchas bacteria, plants, fungi, or animal cells or tissues, naturallyoccurring or synthetic compositions.

Depending upon the particular embodiment being practiced, the testcompounds can be provided free in solution, or can be attached to acarrier, or a solid support, e.g., beads. A number of suitable solidsupports can be employed for immobilization of the test compounds.Examples of suitable solid supports include agarose, cellulose, dextran(commercially available as, i.e., Sephadex, Sepharose) carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper,nitrocellulose, ion exchange resins, plastic films,polyaminemethylvinylether maleic acid copolymer, glass beads, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc.Additionally, for the methods described herein, test compounds can bescreened individually, or in groups. Group screening is particularlyuseful where hit rates for effective test compounds are expected to below such that one would not expect more than one positive result for agiven group.

A number of small molecule libraries are known in the art andcommercially available. These small molecule libraries can be screenedusing the screening methods described herein. A chemical library orcompound library is a collection of stored chemicals that can be used inconjunction with the methods described herein to screen candidate agentsfor a particular effect. A chemical library comprises informationregarding the chemical structure, purity, quantity, and physiochemicalcharacteristics of each compound. Compound libraries can be obtainedcommercially, for example, from Enzo Life Sciences™, Aurora FineChemicals™ Exclusive Chemistry Ltd.™, ChemDiv, ChemBridge™, TimTecInc.™, AsisChem™, and Princeton Biomolecular Research™, among others.

Without limitation, the compounds can be tested at any concentrationthat can exert an effect on the cells relative to a control over anappropriate time period. In some embodiments, compounds are tested atconcentrations in the range of about 0.01 nM to about 100 mM, about 0.1nM to about 500 μM, about 0.1 μM to about 20 μM, about 0.1 μM to about10 μM, or about 0.1 μM to about 5 μM.

The compound screening assay can be used in a high through-put screen.High through-put screening is a process in which libraries of compoundsare tested for a given activity. High through-put screening seeks toscreen large numbers of compounds rapidly and in parallel. For example,using microtiter plates and automated assay equipment, a laboratory canperform as many as 100,000 assays per day in parallel.

The compound screening assays described herein can involve more than onemeasurement of the cell or reporter function (e.g., measurement of morethan one parameter and/or measurement of one or more parameters atmultiple points over the course of the assay). Multiple measurements canallow for following the biological activity over incubation time withthe test compound. In one embodiment, the reporter function is measuredat a plurality of times to allow monitoring of the effects of the testcompound at different incubation times.

The screening assay can be followed by a subsequent assay to furtheridentify whether the identified test compound has properties desirablefor the intended use. For example, the screening assay can be followedby a second assay selected from the group consisting of measurement ofany of: bioavailability, toxicity, or pharmacokinetics, but is notlimited to these methods.

Kits

Another aspect of the technology described herein relates to kits fortreating a lung disease or disorder, kits for screening a candidateagent and/or kits for differentiating a human stem cell to a human lungprogenitor cell or for differentiating a human lung progenitor cell to aspecific type or types of human lung cell(s). Described herein are kitcomponents that can be included in one or more of the kits describedherein.

In one embodiment, the kits described herein can include a human lungprogenitor cell, as that term is used herein. In one embodiment, one ormore signaling pathway agonists or antagonists that promotedifferentiation of a stem cell are included in the kit. In anotherembodiment, a component described herein such as one or more TGF-βreceptor inhibitor(s), one or more BMP agonists, one or more FGFagonists, and instructions for converting a stem cell (e.g., embryonicstem cell, isolated pluripotent stem cell, anterior foregut endodermcell, or definitive endoderm cell) to a human lung progenitor cell,e.g., using a method described herein.

Another aspect of the technology disclosed herein relates to kits toproduce human lung progenitor cells according to the methods asdisclosed herein. In some embodiments, the components described hereincan be provided singularly or in any combination as a kit. The kitincludes the components described herein, e.g., a composition(s) thatincludes a compound(s) described herein, e.g., a compound or cocktail ofcompounds or reagents for differentiating a human stem cell to a lungprogenitor cell. Such kits can optionally include one or more agentsthat permit the detection of a lung progenitor cell marker or a lungcell marker or set thereof. In addition, the kit optionally comprisesinformational material.

In some embodiments, the compound in the kit can be provided in awatertight or gas tight container which in some embodiments issubstantially free of other components of the kit. For example, asignaling pathway or differentiation pathway modulating compound can besupplied in more than one container, e.g., it can be supplied in acontainer having sufficient reagent for a predetermined number ofdifferentiation reactions, e.g., 1, 2, 3 or greater. One or morecompound as described herein can be provided in any form, e.g., liquid,dried or lyophilized form. It is preferred that the compound(s)described herein are substantially pure and/or sterile. When the one ormore signaling pathway modulating compounds described herein is providedin a liquid solution, the liquid solution preferably is an aqueoussolution, with a sterile aqueous solution being preferred. When acompound described herein is provided as a dried form, reconstitutiongenerally is by the addition of a suitable solvent. The solvent, e.g.,sterile water or buffer, can optionally be provided in the kit.

The informational material can be descriptive, instructional, marketingor other material that relates to the methods described herein and/orthe use of a compound(s) described herein for the methods describedherein. The informational material of the kits is not limited in itsform. In one embodiment, the informational material can includeinformation about production of the compound, molecular weight of thecompound, concentration, date of expiration, batch or production siteinformation, and so forth. In one embodiment, the informational materialrelates to methods for using or administering the compound.

In one embodiment, the informational material can include instructionsto administer a human lung progenitor cell as described herein in asuitable manner to effect treatment of a lung injury or a lung diseaseor disorder, e.g., in a suitable dose, dosage form, or mode ofadministration (e.g., a dose, dosage form, or mode of administrationdescribed herein). In another embodiment, the informational material caninclude instructions for differentiating a human stem cell to a humanlung progenitor cell. Alternatively, the informational material caninclude instructions for screening a candidate agent for treating a lungdisease or disorder.

In addition to a compound(s) described herein, the composition of thekit can include other ingredients, such as a solvent or buffer, astabilizer, a preservative, and/or an additional agent, e.g., fordifferentiating stem cells (e.g., in vitro) or for treating a conditionor disorder described herein. Alternatively, the other ingredients canbe included in the kit, but in different compositions or containers thana cell or signaling pathway or differentiation pathway modulatingcompound described herein. In such embodiments, the kit can includeinstructions for admixing a compound(s) described herein and the otheringredients, or for using a compound(s) described herein together withthe other ingredients, e.g., instructions on combining the two agentsprior to use or administration.

The kit can include a component for the detection of a marker for humanlung progenitor cells, ES cells iPS cells, thyroid lineage cells,neuronal lineage cells etc. In addition, the kit can include one or moreantibodies that bind a cell marker, or primers for an RT-PCR or PCRreaction, e.g., a semi-quantitative or quantitative RT-PCR or PCRreaction. Such components can be used to assess the activation of lungcell-specific markers or the loss of ES cell, iPSC, thyroid lineage, orneuronal lineage markers. If the detection reagent is an antibody, itcan be supplied in dry preparation, e.g., lyophilized, or in a solution.The antibody or other detection reagent can be linked to a label, e.g.,a radiological, fluorescent (e.g., GFP) or colorimetric label for use indetection. If the detection reagent is a primer, it can be supplied indry preparation, e.g., lyophilized, or in a solution.

The kit can also include one or more reagents for enhancing theefficiency of induced pluripotent stem cell production, such as an HDACinhibitor (e.g., valproic acid) or a DNA methyltransferase inhibitor(e.g., 5azaC).

In one embodiment, the kit comprises a cell or tissue medium fordefinitive endoderm generation. In one embodiment, the medium comprisesB27, Activin A and ZSTK474. An exemplary definitive endoderm generationmedium comprises 2% B27, 20 ng/mL Activin A, and 0.2-0.5 μM ZSTK474.

The kit will typically be provided with its various elements included inone package, e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g.,a Styrofoam box. The enclosure can be configured so as to maintain atemperature differential between the interior and the exterior, e.g., itcan provide insulating properties to keep the reagents at a preselectedtemperature for a preselected time.

The present invention may be as defined in any one of the followingnumbered paragraphs.

-   1. An isolated human Nkx2.1 positive, Sox2 positive proximal airway    multipotent progenitor cell.-   2. The isolated cell of paragraph 1, wherein the cell is Tuj1    negative and Pax8 negative.-   3. The isolated cell of paragraph 1, wherein the cell is    proliferative and differentiates into an airway basal stem cell, a    ciliated cell, a Clara cell, a neuroendocrine cell, or a squamous    epithelial cell under chosen differentiation conditions.-   4. An isolated human Nkx2.1 positive, Sox9 positive, distal    multipotent lung progenitor cell.-   5. The isolated cell of paragraph 4, wherein the cell is FoxP2    positive and/or ID2 positive.-   6. The isolated cell of paragraph 4, wherein the cell is    proliferative and differentiates into any epithelial lung cell when    placed under chosen differentiation conditions.-   7. The isolated cell of paragraph 6, wherein the cell differentiates    into an airway basal stem cell, a ciliated cell, a Clara cell, a    mucin secreting goblet cell, a type I pneumocyte, a type II    pneumocyte, a squamous epithelial cell, a bronchioalveolar stem    cell, a bronchioalveolar duct junction stem cell, a migratory CK14+    cell, or a neuroendocrine cell when placed under chosen    differentiation conditions.-   8. An isolated human Nkx2.1 positive, p63 positive multipotent    airway basal stem cell.-   9. The isolated cell of paragraph 8, wherein the cell is    proliferative and differentiates into a ciliated cell, a Clara cell,    a mucin secreting goblet cell, or a basal cell when placed under    chosen differentiation conditions.-   10. The isolated cell of paragraph 8, wherein the multipotent airway    basal stem cell does not express a Clara cell marker, a ciliated    cell marker, a neuroendocrine cell marker, or a squamous cell    marker.-   11. The isolated cell of paragraph 8, wherein the cell is Sox2    positive.-   12. The isolated cell of paragraph 8, wherein the cell is CK5    positive and/or NGFR positive.-   13. The isolated cell of paragraph 1, 5, or 8, wherein the cell is a    disease-specific cell.-   14. A composition comprising an isolated human Nkx2.1 positive, Sox2    positive proximal airway multipotent progenitor cell and a scaffold.-   15. The composition of paragraph 14, wherein the scaffold is    implantable in a subject.-   16. The composition of paragraph 14, wherein the cell is autologous    to the subject into which the composition is being implanted.-   17. The composition of paragraph 14, wherein the scaffold is    biodegradable.-   18. The composition of paragraph 14, wherein the scaffold comprises    a natural fiber, a synthetic fiber, decellularized lung tissue, or a    combination thereof.-   19. The composition of paragraph 18, wherein the natural fiber is    selected from the group consisting of collagen, fibrin, silk,    thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and    gelatin.-   20. The composition of paragraph 19, wherein the synthetic fiber is    selected from the group consisting of: representative bio-degradable    aliphatic polyesters such as polylactic acid (PLA), polyglycolic    acid (PGA), poly(D,L-lactide-co-glycolide) (PLGA),    poly(caprolactone), diol/diacid aliphatic polyester,    polyester-amide/polyester-urethane, poly(valerolactone),    poly(hydroxyl butyrate), polybutylene terephthalate (PBT),    polyhydroxyhexanoate (PHH), polybutylene succinate (PBS), and    poly(hydroxyl valerate).-   21. The composition of paragraph 14, wherein the proximal airway    multipotent progenitor cell is Tuj1 negative and/or Pax8 negative.-   22. A composition comprising an isolated human Nkx2.1 positive, Sox9    positive, distal multipotent lung progenitor cell and a scaffold.-   23. The composition of paragraph 22, wherein the multipotent lung    progenitor cell is FoxP2 positive and/or ID2 positive.-   24. The composition of paragraph 22, wherein the scaffold is    implantable in a subject.-   25. The composition of paragraph 22, wherein the cell is autologous    to the subject into which the composition is being implanted.-   26. The composition of paragraph 22, wherein the scaffold is    biodegradable.-   27. The composition of paragraph 22, wherein the scaffold comprises    a natural fiber, a synthetic fiber, decellularized lung, or a    combination thereof.-   28. The composition of paragraph 27, wherein the natural fiber is    selected from the group consisting of collagen, fibrin, silk,    thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and    gelatin.-   29. The composition of paragraph 28, wherein the synthetic fiber is    selected from the group consisting of: representative bio-degradable    aliphatic polyesters such as polylactic acid (PLA), polyglycolic    acid (PGA), poly(D,L-lactide-co-glycolide) (PLGA),    poly(caprolactone), diol/diacid aliphatic polyester,    polyester-amide/polyester-urethane, poly(valerolactone),    poly(hydroxyl butyrate), polybutylene terephthalate (PBT),    polyhydroxyhexanoate (PHH), polybutylene succinate (PBS), and    poly(hydroxyl valerate).-   30. A composition comprising an isolated human Nkx2.1 positive, p63    positive multipotent airway basal stem cell and a scaffold.-   31. The composition of paragraph 30, wherein the airway basal stem    cell is CK5 positive and/or NGFR positive.-   32. The composition of paragraph 30, wherein the scaffold is    implantable in a subject.-   33. The composition of paragraph 32, wherein the cell is autologous    to the subject into which the composition is being implanted.-   34. The composition of paragraph 30, wherein the scaffold is    biodegradable.-   35. The composition of paragraph 30, wherein the scaffold comprises    a natural fiber, a synthetic fiber, decellularized lung tissue, or a    combination thereof.-   36. The composition of paragraph 35, wherein the natural fiber is    selected from the group consisting of collagen, fibrin, silk,    thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and    gelatin.-   37. The composition of paragraph 36, wherein the synthetic fiber is    selected from the group consisting of: representative bio-degradable    aliphatic polyesters such as polylactic acid (PLA), polyglycolic    acid (PGA), poly(D,L-lactide-co-glycolide) (PLGA),    poly(caprolactone), diol/diacid aliphatic polyester,    polyester-amide/polyester-urethane, poly(valerolactone),    poly(hydroxyl butyrate), polybutylene terephthalate (PBT),    polyhydroxyhexanoate (PHH), polybutylene succinate (PBS), and    poly(hydroxyl valerate).-   38. The composition of paragraph 14, 22 or 30, wherein the cell is a    disease-specific cell.-   39. A method of generating a human lung progenitor cell or    population of human lung progenitor cells that is Nkx2.1 positive,    Tuj1 negative and Pax8 negative, the method comprising contacting a    human foregut endoderm cell with FGF2, WNT and BMP4, each for a time    and at a concentration sufficient to permit differentiation of said    human foregut endoderm cell to an Nkx2.1 positive, Tuj1 negative,    Pax8 negative lung progenitor cell.-   40. The method of paragraph 39, wherein the contacting step is    performed for at least 2 days.-   41. The method of paragraph 39, further comprising contacting the    Nkx2.1 positive, Tuj1 negative, Pax8 negative lung progenitor cell    with BMP7, FGF7, a WNT antagonist, and a MAPKK/ERK antagonist, each    for a time and at a concentration sufficient to permit    differentiation of the Nkx2.1 positive, Tuj1 negative, Pax8 negative    lung progenitor cell to a Nkx2.1 positive, Sox2 positive proximal    airway multipotent progenitor cell.-   42. The method of paragraph 41, wherein the Wnt antagonist comprises    IWR-1.-   43. The method of paragraph 41, wherein the MAPKK/ERK antagonist    comprises PD98059.-   44. The method of paragraph 41, wherein the contacting step is    performed for at least 4 days.-   45. The method of paragraph 41, further comprising contacting the    Nkx2.1 positive, Tuj1 negative, Pax8 negative lung progenitor cell    with BMP7, FGF7, a WNT antagonist, and a MAPKK/ERK antagonist each    for a time and at a concentration sufficient to permit    differentiation of the Nkx2.1 positive, Tuj1 negative, Pax8 negative    lung progenitor cell to an Nkx2.1 positive, Sox9 positive distal    multipotent lung progenitor cell.-   46. The method of paragraph 45, wherein the Wnt antagonist comprises    IWR-1.-   47. The method of paragraph 45, wherein the MAPKK/ERK antagonist    comprises PD98059.-   48. The method of paragraph 45, wherein the contacting step is    performed for at least 4 days.-   49. The method of paragraph 39, wherein the culture of foregut    endoderm cells are derived from embryonic stem (ES) cells or induced    pluripotent stem cells (iPSCs).-   50. The method of paragraph 39, further comprising contacting a    culture of Nkx2.1 positive, Tuj1 negative, Pax8 negative cells with    B27, BMP7, FGF7, and a WNT antagonist, each for a time and at a    concentration sufficient to permit differentiation of said Nkx2.1    positive, Tuj1 negative, Pax8 negative lung progenitor cell to an    Nkx2.1 positive, p63 positive multipotent airway basal stem cell.-   51. The method of paragraph 39, further comprising contacting the    culture of Nkx2.1 positive, Tuj1 negative, Pax8 negative cells with    Noggin.-   52. The method of paragraph 50, wherein the contacting step is    performed for at least 10 days.-   53. A method for treating a lung disease or disorder, or lung injury    in a subject, the method comprising: administering a composition    comprising an isolated human lung progenitor cell and a    pharmaceutically acceptable carrier to a subject having a lung    disease or disorder, or lung injury.-   54. The method of paragraph 53, wherein the isolated human lung    progenitor cell is selected from the group consisting of: an Nkx2.1    positive, Sox2 positive proximal airway multipotent progenitor cell;    an Nkx2.1 positive, Sox9 positive distal multipotent lung progenitor    cell, an Nkx2.1 positive, p63 positive multipotent airway basal stem    cell, or a differentiated cell thereof.-   55. The method of paragraph 53, wherein the proximal airway    multipotent progenitor cell is Tuj1 negative and/or Pax8 negative.-   56. The method of paragraph 53, wherein the distal multipotent lung    progenitor cell is FoxP2 and/or ID2 positive.-   57. The method of paragraph 53, wherein the airway basal stem cell    is CK5 positive and/or NGFR positive.-   58. The method of paragraph 53, wherein the composition is    administered to the lung.-   59. The method of paragraph 53, wherein the isolated human lung    progenitor cell is autologous to the subject to which the    composition is being administered.-   60. The method of paragraph 53, wherein the composition further    comprises a scaffold.-   61. The method of paragraph 60, wherein the scaffold is implantable    in a subject.-   62. The method of paragraph 60, wherein the scaffold is    biodegradable.-   63. The method of paragraph 60, wherein the scaffold comprises a    natural fiber, a synthetic fiber, decellularized lung tissue, or a    combination thereof.-   64. The method of paragraph 60, wherein the scaffold comprises an    agent that promotes differentiation of the isolated human lung    progenitor cell.-   65. The method of paragraph 60, wherein the composition is    formulated for aerosol delivery.-   66. A method of screening for an agent to treat a lung disease or    disorder, the method comprising:

(a) culturing a population of human disease-specific airway cellsproduced by in vitro differentiation of a human disease specific lungprogenitor cell in the presence and absence of a candidate agent fortreating a lung disease or disorder,

(b) comparing the expression or activity of at least one marker that isupregulated in the disease or comparing the expression or activity of atleast one marker that is downregulated in the disease in the presenceand absence of the candidate agent,

wherein a decrease in the expression or activity of at least oneupregulated disease marker or an increase in the expression or activityof at least one downregulated disease marker identifies the candidateagent as a candidate for the treatment of the lung disease or disorderin a subject.

-   67. The method of paragraph 66, wherein the method further comprises    steps before step (a) of differentiating a population of isolated    human disease-specific lung progenitor cells to a culture of human    disease-specific airway cells.-   68. The method of paragraph 66, wherein the method further comprises    steps before step (a) of differentiating a population of induced    pluripotent stem cells derived from a subject having a lung disease    or disorder to a population of isolated human disease-specific lung    progenitor cells.-   69. The method of paragraph 66, wherein the candidate agent    comprises a small molecule, a protein, a polypeptide, an antibody or    an antigen binding fragment thereof, or a nucleic acid.-   70. The method of paragraph 66, wherein the human lung progenitor    cell is selected from the group consisting of: a human Nkx2.1    positive, Sox2 positive proximal airway multipotent progenitor cell,    a human Nkx2.1 positive, Sox9 positive distal multipotent lung    progenitor cell, and a human Nkx2.1 positive, p63 positive    multipotent airway basal stem cell.-   71. The method of paragraph 70, wherein the human Nkx2.1 positive,    Sox2 positive proximal airway multipotent progenitor cell, and the    human Nkx2.1 positive, Sox9 positive distal multipotent lung    progenitor cell are made by a method comprising contacting an Nkx2.1    positive, Tuj1 negative, Pax8 negative lung progenitor cell with    BMP7, FGF7, a WNT antagonist, and a MAPKK/ERK antagonist each for a    time and at a concentration sufficient to permit the Nkx2.1    positive, Tuj1 negative, Pax8 negative lung progenitor cell to    differentiate to an Nkx2.1 positive, Sox9 positive proximal airway    multipotent progenitor cell, or to an Nkx2.1 positive, Sox9 positive    distal multipotent lung progenitor cell.-   72. The method of paragraph 71, wherein the contacting step is    performed for at least 4 days.-   73. The method of paragraph 70, wherein the human Nkx2.1 positive,    p63 positive multipotent airway basal stem cell is made by a method    comprising contacting an Nkx2.1 positive, Tuj1 negative, Pax8    negative cell with B27, BMP7, FGF7, and a WNT antagonist, each for a    time and at a concentration sufficient to permit differentiation of    said Nkx2.1 positive, Tuj1 negative, Pax8 negative lung progenitor    cell to an Nkx2.1 positive, p63 positive multipotent airway basal    stem cell.-   74. The method of paragraph 73, further comprising contacting the    Nkx2.1 positive, Tuj1 negative, Pax8 negative cell with Noggin.-   75. The method of paragraph 73, wherein the contacting step is    performed for at least 10 days.-   76. A method of screening for an agent to induce differentiation of    a human lung progenitor cell, the method comprising:

(a) culturing an Nkx2.1 positive, Tuj1 negative, Pax8 negative humanlung progenitor cell in the presence and absence of a candidatedifferentiation agent,

(b) comparing the expression or activity of at least one marker that isupregulated during differentiation of the lung progenitor cell to a moredifferentiated state or comparing the expression or activity of at leastone marker that is downregulated during differentiation of the lungprogenitor cell to a more differentiated state in the presence andabsence of the candidate agent,

wherein a decrease in the expression or activity of at least oneupregulated differentiation marker or an increase in the expression oractivity of at least one downregulated differentiation marker isindicative that the candidate agent can be used to inducedifferentiation of an isolated human lung progenitor cell in a subject.

-   77. The method of paragraph 76, wherein the method further comprises    steps before step (a) of differentiating an embryonic stem cell or    induced pluripotent stem cell to a human lung progenitor cell.-   78. The method of paragraph 76, wherein the candidate agent    comprises a small molecule, a protein, a polypeptide, an antibody,    or a nucleic acid.-   79. The method of paragraph 76, wherein the human lung progenitor    cell is selected from the group consisting of: a human Nkx2.1    positive, Sox2 positive proximal airway multipotent progenitor cell,    a human Nkx2.1 positive, Sox9 positive distal multipotent lung    progenitor cell, and a human Nkx2.1 positive, p63 positive    multipotent airway basal stem cell.-   80. The method of paragraph 79, wherein the human Nkx2.1 positive,    Sox2 positive proximal airway multipotent progenitor cell, and the    human Nkx2.1 positive, Sox9 positive distal multipotent lung    progenitor cell are made by a method comprising contacting an Nkx2.1    positive, Tuj1 negative, Pax8 negative human lung progenitor cell    with BMP7, FGF7, a WNT antagonist, and a MAPKK/ERK antagonist each    for a time and at a concentration sufficient to permit the Nkx2.1    positive, Tuj1 negative, Pax8 negative lung progenitor cell to    differentiate to an Nkx2.1 positive, Sox9 positive proximal airway    multipotent progenitor cell, or to an Nkx2.1 positive, Sox9 positive    distal multipotent lung progenitor cell.-   81. The method of paragraph 80, wherein the contacting step is    performed for at least 4 days.-   82. The method of paragraph 79, wherein the human Nkx2.1 positive,    p63 positive multipotent airway basal stem cell is made by a method    comprising contacting an Nkx2.1 positive, Tuj1 negative, Pax8    negative cell with B27, BMP7, FGF7, and a WNT antagonist, each for a    time and at a concentration sufficient to permit differentiation of    the Nkx2.1 positive, Tuj1 negative, Pax8 negative lung progenitor    cell to an Nkx2.1 positive, p63 positive multipotent airway basal    stem cell.-   83. The method of paragraph 82, further comprising contacting the    Nkx2.1 positive, Tuj1 negative, Pax8 negative cell with Noggin.-   84. The method of paragraph 82, wherein the contacting step is    performed for at least 10 days.-   85. A kit for treating a lung disease or disorder, the kit    comprising: a cell of paragraph 1, 5, or 9, a pharmaceutically    acceptable carrier, and instructions for treating a lung disease or    disorder.-   86. The kit of paragraph 85, further comprising a scaffold.-   87. A kit for screening a candidate agent, the kit comprising: a    cell of paragraph 1, 5, or 8, one or more agents for detecting lung    specific cell surface markers, and instructions therefore.-   88. The kit of paragraph 87, further comprising a cell culture    medium, a growth factor, and/or a differentiation agent.-   89. A kit for differentiating a human stem cell to a human lung    progenitor cell, the kit comprising:-   (i) two or more of BMP4, FGF2, WNT, BMP7, FGF7, a WNT antagonist, a    PI3 kinase inhibitor, Activin A, Noggin, B27, and retinoic acid,    optionally provided in unit doses;-   (ii) optionally, a cell culture medium;-   (iii) one or more agents for detecting a lung cell-specific surface    marker; and-   (iii) instructions therefore.-   90. A cell or tissue medium comprising: B27, Activin A, and ZSTK474.-   91. The medium of paragraph 90, wherein the concentration of B27 is    1%-5%.-   92. The medium of paragraph 91, wherein the concentration of B27 is    2%.-   93. The medium of paragraph 90, wherein the concentration of Activin    A is 10-40 ng/mL.-   94. The medium of paragraph 93, wherein the concentration of Activin    A is 20 ng/mL.-   95. The medium of paragraph 90, wherein the concentration of ZSTK474    is 0.2-0.5 μM.-   96. The medium of paragraph 90, wherein the medium comprises the    components of RPMI medium.-   97. A cell or tissue medium comprising: CHIR9902, PIK-75,    Dorsomorphin, and FGF2.-   99. The medium of paragraph 97, wherein the concentration of    CHIR9902 is in the range of 0.1-1 μM.-   100. The medium of paragraph 97, wherein the concentration of PIK-75    comprises 0.01-0.1 μM.-   101. The medium of paragraph 97, wherein the concentration of    dorsomorphin comprises 1-5 μM.-   102. The medium of paragraph 97, wherein the concentration of FGF2    comprises 10-100 ng/mL.-   103. The medium of paragraph 97, further comprising a drug selected    from the group consisting of: GF-109203X, Ro31-8220, Pp242, PIK-75,    ZSTK474, PMA, carvedilol, corticosterone, triclabendazole,    benproperine phosphate, phenothiazine, and methotrexate.-   104. A method of generating a human lung progenitor cell or    population of human lung progenitor cells that is Nkx2.1 positive,    Tuj1 negative, and Pax8 negative, the method comprising contacting a    human foregut endoderm cell with a Wnt agonist, a PIK3 kinase    inhibitor, a BMP antagonist, and a drug selected from the group    consisting of GF-109203X, Ro31-8220, Pp242, PIK-75, ZSTK474, PMA,    carvedilol, corticosterone, triclabendazole, benproperine phosphate,    phenothiazine, and methotrexate, each for a time and at a    concentration sufficient to permit differentiation of said human    foregut endoderm cell to an Nkx2.1 positive, Tuj1 negative, Pax8    negative lung progenitor cell.-   105. The method of paragraph 104, wherein the contacting step is    performed for at least 2 days.-   106. The method of paragraph 104, wherein the Wnt agonist comprises    CHIR9902.-   107. The method of paragraph 106, wherein the concentration of    CHIR9902 is in the range of 0.1-1 μM.-   108. The method of paragraph 104, wherein the PI3 kinase inhibitor    comprises PIK-75.-   109. The method of paragraph 108, wherein the concentration of    PIK-75 comprises 0.01-0.1 μM.-   110. The method of paragraph 104, wherein the BMP antagonist    comprises Dorsomorphin.-   111. The method of paragraph 110, wherein the concentration of    dorsomorphin comprises 1-5 μM.-   112. The method of paragraph 104, wherein the growth factor    comprises FGF2.-   113. The method of paragraph 112, wherein the concentration of FGF2    comprises 10-100 ng/mL.-   114. A method for generating a definitive endoderm cell or    population of definitive endoderm cells, the method comprising    contacting an iPSC or ESC with B27, Activin A, and ZSTK474, each for    a time and at a concentration sufficient to permit differentiation    of the iPSC or ESC to a definitive endoderm cell.-   115. The method of paragraph 114, wherein the generation of a    definitive endoderm cell is determined by FOXA2/SOX17 co-staining or    by FACS analysis with cKit/CXCR4 and/or cKit/EpCAM combination.-   116. The method of paragraph 114, wherein the concentration of B27    is 1%-5%.-   117. The method of paragraph 116, wherein the concentration of B27    is 2%.-   118. The method of paragraph 114, wherein the concentration of    Activin A is 10-40 ng/mL.-   119. The method of paragraph 118, wherein the concentration of    Activin A is 20 ng/mL.-   120. The method of paragraph 114, wherein the concentration of    ZSTK474 is 0.2-0.5 μM.

EXAMPLES

The discovery of embryonic stem cells (ES cells) and induced pluripotentstem cells (iPSCs) has resulted in an unprecedented opportunity toproduce tissue-specific cell types that can be used in human diseasemodeling, drug screening, and patient-specific therapies. Although manyiPSCs from patients with lung diseases are currently being produced, themajor obstacle preventing the actual development of human lung diseasemodels using these cells is the inability to convert them into lungprogenitors and then subsequently into differentiated pulmonaryepithelial cell types for therapeutic treatment or to study theircharacteristics in the laboratory. Several attempts to produce pulmonaryepithelial cells from mouse and human ES cells have been made. In manycases, investigators have focused on the generation of type IIpneumocytes (Van Vranken et al., 2005; Samadikuchaksaraei et al., 2006;Wang et al., 2007). Fewer studies have targeted the differentiation ofairway epithelial cells from pluripotent stem cells despite the factthat airway diseases such as asthma, Cystic Fibrosis, bronchitis andbronchogenic carcinoma are, in aggregate, more prevalent than thediseases of the alveoli such as emphysema. Furthermore, when the lungprimordium forms, the first two recognizable progenitors are themultipotent embryonic lung progenitors and the airway progenitors. Theproduction of these two progenitors is expected, therefore, to permitthe differentiation of each type of lung epithelial cell.

The large airway epithelium of the mouse and the majority of the humanairway epithelium is comprised of four major cell types: basal stemcells abutting the basement membrane, secretory detoxifying Clara cellswith P450 machinery primarily in the mouse, ciliated cells that propelmucous out of the respiratory tree, and goblet cells that respond toinjury or inflammation and secrete the mucous. Prior attempts (Coraux etal., 2005; Van Haute et al., 2009) to generate functional pulmonaryepithelium from ES cells were characterized by the stochastic productionof limited numbers of cells and the generation of mixed cell populationsthat contain undifferentiated pluripotent stem cells that carry asignificant risk of teratoma formation after transplantation. Othershave utilized incompletely defined media to induce airway celldifferentiation (such as the exposure of human ES cells to tumor cellextract; Roszell et al., 2009) or used genetically modified pluripotentstem cells that were selected based upon the presence of a drugresistance gene (Wang et al., 2007). Unfortunately, genetic modulationraises the possibility of the introduction of deleterious geneticmutations in the resulting cells.

Given the difficulty of obtaining adult human airway stem cells withwhich to model human lung disease, the studies described herein seek togenerate normal and disease-specific lung epithelial cells from humannormal and lung disease-specific iPSCs. This is particularly importantbecause murine models of lung disease often do not phenocopy human lungdisease. A prime example is the Cftr knockout mouse that does notdisplay the Cystic Fibrosis disease-associated lung pathology observedin human patients (Snouwaert et al., 1992; Clarke et al., 1992;Guilbault et al., 2006). Furthermore, iPSC-derived epithelial cells willreflect the pre-pathologic state of lung cells in which multiplesecondary changes, such as those associated with inflammation orinfection, do not obscure the effects of the initial genetic abnormalityin disease pathogenesis.

The overall strategy described herein employs a step-wisedifferentiation approach. Mechanisms that regulate mouse embryonicendoderm regionalization, lung specification and subsequent progenitorpatterning and growth have been well studied (reviewed in Morrisey andHogan, 2010). The onset of lung specification within the endoderm isaccompanied by Nkx2.1 expression and the downregulation of Sox2 alongthe dorsal-ventral axis of the gut tube (Lazzaro et al., 1991; Minoo etal., 1999; Que et al., 2009). Nkx2.1 is the earliest marker of lungendoderm distinguishing it from the rest of the foregut endoderm. Later,Sox2 expression again increases in the area of the future trachea,bronchus and bronchioles while Sox9, FoxP2 and ID2 remain in the distallung bud tip and are markers of a multipotent embryonic lung progenitorpopulation (Perl et al., 2005; Shu et al., 2007; Rawlins et al., 2009).Then, Nkx2.1+Sox2+ airway progenitor cells located in embryonic tracheadifferentiate into p63+ airway basal stem cells (Que et al., 2009). Themouse large airway epithelium is highly reminiscent of the majority ofhuman airway epithelium (Rock et al., 2010; Rock et al., 2009; Evans etal., 2001) and can, therefore, be used as a model to study human airwaydisease.

The inventors focused on producing Nkx2.1+ lung endoderm that is devoidof Nkx2.1+ thyroid endoderm and neural progenitors. A recent reportusing human ES cells demonstrated that a differentiation strategy basedon mimicking mouse gut organogenesis led to the production of anteriorforegut cells and Nkx2.1+ cells with efficiencies up to 30% (Green etal., 2011). However, these Nkx2.1+ cells were not demonstrated to becomposed of purely lung progenitors. TUJ1 (a marker of neuronal tissue)and PAX8 (a marker of thyroid tissue) expression was not evaluated atthe single cell level by co-antibody staining with an Nkx2.1 antibody.Development of a novel strategy to produce Nkx2.1+ progenitors thatreflect only lung and not thyroid or brain differentiation is thereforeneeded, and the technique must be applicable to human disease-specificiPSCs in order for the technique to become widely relevant to the studyof human lung disease.

Described herein is an efficient and consistent step-wisedifferentiation method to generate definitive endoderm (DE), foregutendoderm, early lung endoderm, multipotent Nkx2.1+ embryonic lungprogenitor cells and airway progenitor cells beginning with mouse EScells (FIG. 1) and subsequently with normal and lung disease-specifichuman iPSCs. The inventors show that a high dose of Activin, transientWNT activation and staged BMP4 inhibition converts ES cells intodefinitive endoderm with high efficiencies. Further inhibition of TGFβalone is sufficient to anteriorize endoderm into Sox2+ foregut endoderm.These studies indicate that the anteriorization of the endoderm toforegut endoderm enhances the ultimate differentiation of Nkx2.1+ cellsat later steps of the strategy. BMP4, FGF2 and WNT are each necessaryfor induction of the Nkx2.1+ immature lung progenitors. These earlyprogenitors can mature into Nkx2.1+Sox2+ proximal progenitor cells andNkx2.1+p63+ airway basal stem cells in vitro, and can differentiate intomature airway epithelium.

Example 1A: A Highly Efficient, Universally Applicable Protocol forImproving the Efficiency of Definitive Endoderm Production

The endoderm is one of the three primary germ cell layers in the embryo.It forms the embryonic gut which in turn gives rise to the epithelia ofthe lungs and digestive organs. Therefore, the first essential step fordirecting human iPSC towards a lung lineage is to generate endodermcells, preferably with high efficiency. For a variety of reasons, somecell lines do not generate definitive endoderm with high efficiency. Forexample, the current published protocol (Mou et al., 2012; RPMI+2%B27+100 ng/ml TGF-beta agonist Activin A+5 uM LY-294002 PI3K Inhibitor)is effective in several human iPSC and ESC lines with definitiveendoderm efficiency higher than 90%. In some “difficult” or “resistant”cell lines, this same protocol generates definitive endoderm that variesbetween low efficiency (<30%) to medium efficiency (40-70%). Theinventors screened multiple PI3K inhibitors including LY-294002, ZSTK474(Zenyaku Kogyo Co.™), Wortmannin, PI828 (PIramed Ltd.™, Roche™),NVP-BKM120 (Novartis™), and PIK-75 (Drug Discovery Research, AstellasPharma Inc.™), and determined that ZSTK474 is the most potent fordefinitive endoderm generation. In addition, the use of ZSTK474 permitsthe dose of the growth factor Activin A to be greatly decreased (e.g.,from 100 ng/ml down to 20 ng/ml and in some cell lines down to 10 ng/mlActivin A). Therefore, this protocol is much more cost-effective thanprior protocols. The PI3 kinase inhibitors noted above can also beobtained from commercial sources including, but not limited to,Promega™, Invivogen™, Sigma-Aldrich™, Cayman Chemicals™, Tocris™ andCell Signaling Technologies™.

This protocol permits a remarkable 90% or greater, including 91%, 92%,93%, 94%, 95%, 96%, 97%, and even as high as 98% or more, of humaniPSC/ESC cells to be converted into definitive endoderm cell asdetermined by nuclear staining for the transcription factors Foxa2 andSox17. Importantly, this protocol is very consistent from one experimentto another (i.e., reproducible). Furthermore, this protocol can beuniversally applied to multiple cell lines including several “difficult”cell lines that were determined to have low efficiency of definitiveendoderm generation.

Protocol outline: Human ESC and iPSCs are grown with complete mTeSR1medium on Geltrex™-coated plates (reduced growth factor (RGF) basementmembrane extract (BME) purified from murine Engelbreth-Holm-Swarmtumor). The cells are fed every day and the differentiation is done at65-80% confluence. For definitive endoderm differentiation, mTeSR1 wasaspirated and the cells were rinsed with warm RPMI-1640 two times toremove the residual growth factors in mTeSR1. The medium was replacedwith definitive endoderm differentiation medium (RPMI+2% B27+20 ng/mlActivin A+0.2-0.5 uM ZSTK474). The medium was changed daily over a totaldifferentiation time of 4 days. The definitive endoderm efficiency wasexamined by FOXA2/SOX17 co-staining or by FACS analysis with cKit/CXCR4and cKit/EpCAM combination.

A schematic depicting the protocol for high efficiency definitiveendoderm generation is shown herein in FIG. 13.

Example 1B: Stage-Specific TGFβ Inhibition Regionalizes Naïve Endodermto Anterior Foregut Endoderm and Facilitates the Differentiation ofNkx2.1+ Cells

A recent report described a monolayer-based strategy using synergisticactivation of Nodal and Wnt-β-catenin signaling as well as staged BMP4inhibition to direct mouse ES cells towards an endoderm fate with highefficiency (Sherwood et al., 2011). This technology was adapted hereinto generate definitive endoderm (DE) from mouse ES cells. FIG. 7 shows aschematic strategy and time frame to generate definitive endoderm. Thisprotocol resulted in a remarkable 80%-90% of cells being converted intoDE based on the dual expression of the endoderm transcription factorsFoxA2 and Sox17 (data not shown). Next, it was asked if the newlygenerated DE cells have the competence to generate Nkx2.1+ cells. Afterexposing them for 2 days to serum-free medium containing BMP4, FGF2 andGSK3iXV (WNT agonist) (FIG. 2A), it was observed that less than 1% ofthe cells were Nkx2.1+ (data not shown). However, more than 60% of thecells were Cdx2+, indicating that the majority of the cells werespecified to a hindgut fate (data not shown). Since the endoderm cellsat this stage did not efficiently differentiate into Nkx2.1+ cells, itwas tested whether an “anteriorization” step would facilitate lung fatespecification. Snoeck and colleagues reported that Noggin (a BMPinhibitor) synergized with SB431542 (a TGFβ inhibitor) to suppress aposterior endoderm fate (Cdx2+) in favor of an anterior endoderm fate(Sox2+) (Green et al., 2011). Since the inventors had alreadyincorporated BMP inhibition during DE generation (FIG. 7), the questionwhether continuous BMP inhibition is necessary for anteriorization or ifTGFβ inhibition alone was sufficient for anterior patterning wasexplored. Endoderm was treated with combinations of Activin (a TGFβagonist), A-83-01 (a TGFβ antagonist), BMP4, and Dorsomorphin (a BMP4antagonist) for 2 days (FIG. 2B). The number of FoxA2+Sox2+ anteriorendoderm cells was compared to the total number of FoxA2+ endoderm cells(FIG. 2B). The treated cells were further exposed to the BMP4/FGF2/WNTagonist cocktail for another 2 days to examine their competence togenerate Nkx2.1+ cells (FIG. 2B). The results indicate that TGFβinhibition itself was sufficient to increase FoxA2+Sox2+ anteriorendoderm (40%-55%, compared to <0.1% in medium alone, FIG. 2B) andenhance the competence of endoderm cells to form Nkx2.1+ cells (˜7%-13%as compared to <1% without regionalization, FIG. 2B). It was observedthat continuous Activin treatment resulted in rare FoxA2+Sox2+ cells andfew Nkx2.1+ cells (FIG. 2B), indicating that the optimal duration ofActivin exposure plays a role in the efficiency of regionalization. Itwas also found that additional BMP4 inhibition was not required for thegeneration of FoxA2+Sox2+ cells and ultimately can result in slightlylower percentages of Nkx2.1+ cells (FIG. 2B). It was also observed thatless neuroectodermal marker Tuj1 was present if BMP4 was present betweendays 5-7 (data not shown) in agreement with the finding that BMP4suppresses neural commitment and promotes non-neural lineagedifferentiation from ESCs (Zhang et al., 2010). FGF and WNT signalinghave been shown to maintain hindgut identity and actively repressforegut endoderm differentiation (Wells and Melton, 2000; Ameri et al.,2010). It was tested if their inhibition would enhance anteriorizationtogether with a TGFβ inhibitor. Subsequent experiments revealed an FGFantagonist (PD173074) induced cell death, while a WNT antagonist (IWR-1)did not increase the FoxA2+Sox2+ population (data not shown).

Example 2: Mouse ESC-Derived Nkx2.1+ Cells are Devoid of Neuronal andThyroid Markers, are Proliferative, and Contain Distal Multipotent LungProgenitors and Proximal Airway Progenitors

Nkx2.1 is not a specific marker of the lung, and its expression is alsofound in the thyroid and ventral forebrain. Therefore, it was importantto determine the identity of the Nkx2.1+ cells generated in the culturesystem used herein. Unfortunately, there are no reliable markers of thelung lineage at embryonic stage E9 that are not expressed in the brainor thyroid. Sp-C (Surfactant protein C), which is the most specificmarker for lung epithelial progenitors, is not detected until E10-E11(Wert et al., 1993). SOX9 and FOXP2 are indeed co-expressed with NKX2.1in distal lung multipotent epithelial progenitor cells (data not shown)and are not found in brain or newly specified thyroid (data not shown),but their expression is not evident until after E11-E12. SOX2, on theother hand, is expressed in the E9 lung endoderm (data not shown) andlater in airway epithelial progenitors (data not shown), but SOX2 canalso be found in NKX2.1-expressing cells in ventral forebrain (FIG. 8and data not shown). Thus, at the earliest stage of lung endodermspecification (E8.75-E9), none of these markers (SPC, SOX2, SOX9 andFOXP2) can be used to reliably and uniquely identify lung cells. Incontrast, TUJ1 expression in Nkx2.1+ cells in ventral forebrain beginsat E8.0 and continues to be present thereafter in ventral forebrain andnot in thyroid or lung (FIG. 8 and data not shown), making Tuj1expression a specific indicator of neuronal cell identity within theNKX2.1 domain. PAX8 expression is detected in the primordial thyroid atE8.75 at the time of thyroid specification (data not shown) but not inthe lung or brain. Therefore, PAX8 can be used as a specific indicatorof thyroid cell identity in the NKX2.1 domain (FIG. 8 and data notshown). In summary, the inventors conclude that at the earlyspecification stage, Nkx2.1+/Tuj1−/Pax8− cells can be regarded as havinga lung cell lineage. Accordingly, the ESC-derived Nkx2.1+ cells wereinterrogated for TUJ1 and PAX8 expression to exclude neuronal andthyroid identity (data not shown). As expected for any Nkx2.1+ cell inthe embryo (thyroid, lung and ventral forebrain), all of the ex vivodifferentiated Nkx2.1+ cells co-stained with Foxa2 (data not shown). Asmall subset of Tuj1+ cells were detected in the cultures, but theseneuronal cells did not overlap with the Nkx2.1+ cells (data not shown).The inventors did not detect any Pax8+ cells in their culture (data notshown). Therefore, these data indicate that the Nkx2.1+ cells that aredifferentiated in vitro represent lung endoderm cells. These Nkx2.1+cells are proliferative, with more than half expressing Ki67 (data notshown).

Data from immunofluorescence experiments indicate that ESC-derivedNkx2.1+ cells are devoid of neuronal and thyroid markers, areproliferative, and possess markers of proximal and distal lung endoderm.Immunofluorescence staining for FOXA2, TUJ1 and PAX8 was performedshowing that Nkx2.1+ cells are positive for endodermal marker FOXA2,negative for neuroectodermal marker TUJ1, and negative for thyroidmarker PAX8 (data not shown). Nkx2.1+ cells were proliferative asdemonstrated by co-staining of Nkx2.1 with KI67 (data not shown).Immunofluorescence staining also indicated the presence ofsubpopulations of Nkx2.1+ cells that are positive for SOX2 (an airwayprogenitor marker) and FOXP2/SOX9 (multipotent lung progenitor marker)(data not shown).

In the mouse, Sox2 is rapidly downregulated in the foregut pre-lungendoderm just as Nkx2.1 expression initiates in this same pre-lungendoderm during the process of lung specification at E9 (data notshown). Thus, the very earliest of lung endoderm cells are Nkx2.1+ andSox2-low cells. Soon thereafter, sustained high-level SOX2 expression isdetected in the proximal airway epithelial progenitors of the futuretrachea, bronchus and bronchioles during the process of branchingmorphogenesis (data not shown). Airway progenitors are thereforeNkx2.1+Sox2+.

In contrast, SOX9 and FOXP2 are expressed exclusively in the distal tipmultipotent lung progenitor cells (data not shown), making SOX9 andFOXP2 markers that uniquely identify a distinct population ofmultipotent embryonic lung progenitor cells within the NKX2.1 domainsince they are not present in the proximal airway progenitors or thethyroid or brain as above. Therefore, the inventors checked if their EScell-derived Nkx2.1+ cells at day 9 contained both proximal airway(Nkx2.1+Sox2+) and distal multipotent (Nkx2.1+Sox9+; or Nkx2.1+FoxP2+)progenitor cells. The Nkx2.1+ cells generated from 10 independentexperiments were counted. The average proportion of airway progenitorcells (Nkx2.1+Sox2+) was 1.8%+/−0.8% out of the total Nkx2.1+ cells,while the proportions of distal multipotent progenitor cells,Nkx2.1+Sox9+ or Nkx2.1+FoxP2+, were 4.8%+/−2.5% and 6.6+/−3.1% out oftotal Nkx2.1+ cells, respectively. The remaining Nkx2.1+ cells (notthyroid or neural) are cells with minimal SOX2 expression and canreflect early pre-lung endoderm. Thus, the majority of Nkx2.1+ cells atday 9 likely represent an early lung endoderm that has not beenspecified to an airway progenitor or a distal tip multipotent lungprogenitor population. Immunofluorescence indicates that the cells areenriched in Nkx2.1+Sox2+, Nkx2.1+Sox9+ and Nkx2.1+FoxP2+ cells (data notshown). In aggregate, these results imply that ESC-derived Nkx2.1+ cellsat day 9 were immature, with most lacking proximal and distal progenitorcell markers, representing early lung endoderm cells. Although rare, theexistence of Nkx2.1+Sox2+, Nkx2.1+Sox9+ and Nkx2.1+FoxP2+ cellsindicated that the ES cell-derived Nkx2.1+ lung endoderm cells weredifferentiating into progenitor cells of the airway and multipotent lungprogenitor cells that could later be differentiated into matureepithelial cells.

The expression of lung and other anterior cell fate lineage genesincluding FoxN1 (thymus), Pax8 (thyroid), and Pax9 and Tbx1 (pharyngealpouch endoderm) was analyzed by real-time qPCR (FIG. S4A). As expected,the results showed that Sox2 was downregulated as pluripotent cellsdifferentiated into definitive endoderm (DE). Later, Sox2 levelsincreased in accord with the expectation that anterior endodermexpresses Sox2. Nkx2.1, Sox9 and FoxP2 expression was minimal in ESCs,definitive endoderm and anteriorized endoderm, but was greatly increased(10-20 fold) after stimulation with the FGF2/WNT/BMP4 inductioncocktail, consistent with previous results demonstrating theselung-specific marker combinations by antibody staining (data not shown).Similarly, FoxN1, Tbx1, Pax8, and Pax9 expression was low in ESCs,definitive endoderm and anteriorized endoderm. However, in growthfactor-induced anterior foregut cells, a modest increase in Pax9 andTbx1 expression was observed, whereas FoxN1 and Pax8 expression wasstill very minimal. Despite the increase in Pax9 and Tbx1 geneexpression, the inventors did not detect any reproducible staining forthese proteins by immunofluorescence (data not shown). All these resultsare consistent with the finding that a combination of FGF, BMP4, and WNTsignaling predominantly drives the differentiation of lung-specificNkx2.1+ progenitor cells from anterior endoderm.

Example 3A: BMP4, FGF and WNT Signaling are Each Necessary for NKX2.1Induction In Vitro, Recapitulating Murine Lung Development

It was tested if BMP4, FGF2, and WNT signaling are each required tospecify lung endoderm from the anterior foregut in an in vitro mouse ESCdifferentiation system. To do this, anteriorized endoderm cells wereexposed to combinations of BMP4, FGF2 and WNT agonists and antagonists(FIGS. 3A and B). It was found that BMP4 alone could induce NKX2.1expression. In contrast, FGF2 or/and GSK3i without BMP4 was notsufficient for NKX2.1 induction, indicating that BMP4 signaling isrequired for lung specification (FIGS. 3B and C). Interestingly, in thepresence of FGF and WNT antagonists, BMP4 could no longer induce Nkx2.1+cells. This result implies an endogenous secretion of FGF and WNTligands. It also suggests that FGF and WNT activation are needed forBMP4-dependent Nkx2.1 specification (FIGS. 3B and C). Despitepresumptive endogenous FGF signaling, an added dose of FGF2concentration upregulated Nkx2.1+ cells (FIGS. 3B and C). Compared toFGF2 signaling, exaggerated WNT activation was detrimental, with adecrease in Nkx2.1+ cells (FIGS. 4B and C) and an increase in hindgutCdx2+ cells (data not shown).

BMP2, 4 and 7 are by far the most studied members of the BMP family.Both BMP2 and BMP4 signal through the type I receptor (ALK3), whereasBMP7 binds to a separate type I receptor (ALK2) (reviewed by von Bubnoffand Cho, 2001; Chen et al., 2004; Sieber et al., 2009; Miyazono et al.,2010). The effects of BMP2 and BMP7 were compared to BMP4 on inductionof Nkx2.1+ cells from the anterior endoderm. The results showed thatBMP2 was less efficient than BMP4 at inducing Nkx2.1+ cells, while BMP7(10 ng/ml) had no effect (data not shown). Even with an increasedconcentration (>100 ng/ml), BMP7 still failed to generate Nkx2.1+ cells(data not shown). This indicates that BMP7 signaling via the ALK2receptor is not required for NKX2.1 induction. In canonical BMP2/4signaling, BMP2/4 binds to BMP receptor I/II complex, leading tophosphorylation of Smad1/5/8, followed by formation of heteromericcomplexes with Smad4. These complexes translocate to the nucleus andactivate expression of target genes (von Bubnoff and Cho, 2001; Chen etal., 2004; Sieber et al., 2009; Miyazono et al., 2010). BesidesSmad1/5/8-mediated transcription, BMP-induced receptor complexes canactivate the mitogen-activated protein kinase (MAPK) pathway via ERK,JNK or p38 (Kozawa et al., 2002). Using Dorsomorphin (a pSmad1/5/8inhibitor) and PD98059 (a MAPKK/ERK inhibitor), it was observed thatDorsomorphin completely abrogated generation of Nkx2.1+ cells, whilePD98059 only partially decreased the Nkx2.1+ cell proportion (data notshown). Significant cell death was observed in the presence of PD98059,so the possibility that the decrease in Nkx2.1+ cells was due toapoptosis could not be excluded. In addition, Smad and MAPK-mediatedpathways can be integrated (Aubin et al., 2004), and the decrease inNkx2.1+ cells in the presence of PD98059 might also be due to adownregulation of Smad-dependent signaling. Overall, it was concludedthat Smad-dependent BMP2/4 signaling cascade is necessary for NKX2.1specification from foregut cells (FIG. 4).

Example 3B: Nkx2.1+ Differentiation to Generate Lung Progenitors

A high throughput chemical screen was used to identify compounds thatcan facilitate Nkx2.1+ lung progenitors and also identifies novelsignaling pathways controlling human lung specification.

In order to increase the efficiency of Nkx2.1+ lung progenitorproduction, the inventors performed an unbiased high throughput chemicalscreening on a kinase pathway inhibitor library and an NIH clinical druglibrary to identify small molecule compounds and FDA-approved clinicaldrugs that can facilitate NKX2.1-positive lung progenitordifferentiation. The platform for chemical screening is illustrated inFIG. 14A. The screening was performed at the step of conversion ofFoxa2+Sox2+ anterior endoderm to Nkx2.1+ lung endoderm.

At least five molecules were identified from the Kinase Compound Set(240 compounds) and at least 6 drugs were identified from the NIHClinical Collection (400 compounds) that produced statisticallysignificant (more than 3 fold) increases in the percentage of Nkx2.1+cells (FIG. 14B). These Nkx2.1+ cells have been further stained inseparate experiments to exclude the expression of neuronal lineagemarker Tuj1 and thyroid lineage marker Pax8. In addition, all positivecandidates identified in the primary screen have been assessed for drugduration and dose effects to increase the formation of lung progenitors,and they have been evaluated for other effects including inducingcytotoxicity, and cellular proliferation by Ki67 staining. Since thesecompounds are annotated, the inventors found that there are multiplepositive hits targeting the same pathway such as PI3K and PKC-relatedsignaling pathways. Without wishing to be bound by theory, these dataindicate that these biological pathways are actively involved in humanlung organogenesis. It was also determined that BMP4 is dispensable forhuman lung progenitor differentiation from iPSCs, indicating a speciesdifference in lung development in human and mouse. It was determinedthat inhibition of BMP4 generates lung progenitors with a high NKX2.1expression level, while addition of BMP4 decreases NKX2.1 expressionwithout affecting the total NKX2.1+ lung progenitor cell number.

In addition to positive hits, some compounds were found to decrease oreven block NKX2.1+ cell production. Several of these negative compoundsare MEK1/2 antagonists. Without wishing to be bound by theory, thesedata indicate that MEK1/2-related signaling pathways play importantroles in human lung organogenesis and lung progenitor differentiationfrom iPSCs. For example, endoderm-specific deletion of MEK1/2 results inlung agenesis in mice (unpublished data). In the in vitrodifferentiation systems, the inventors found activation of MEK1/2 (suchas by phorbol 12-myristate 13-acetate or PMA, and other MEK1/2 agonists)stimulates NKX2.1+ cell production.

After studying the additive and synergistic effects of the positivehits, a robust and economically efficient new protocol was devised forthe generation of a highly enriched population of Nkx2.1+ lungprogenitors (up to 85%-90%) from multiple iPS cell lines in xeno-freeconditions in order to keep the cells suitable for future clinicalapplications. This exemplary differentiation medium contains one Wntagonist (e.g., 0.5 uM CHIR9902), one PI3 kinase inhibitor (e.g.,0.02-0.05 uM PIK-75), one clinically used drug (e.g., 0.02-0.05 uMmethotrexate), one BMP antagonist (e.g., 2-4 uM Dorsomorphin) and asingle growth factor (e.g., 10-50 ng/ml FGF2). FIG. 12 is the summary ofthe newly developed step-wise airway progenitor differentiation protocolfrom iPSC to airway progenitors. Many other protocols can be generatedby one of skill in the art from this basic protocol (RPMI+2% B27+10-50ng/ml FGF2+0.5 uM CHIR9902+2-4 uM Dorsomorphin) together with thecompounds listed in FIG. 14.

Based on the current cost of media etc., the inventors calculate thecost of differentiation medium from iPSC to lung progenitors (step I tostep III) is only 10% of the original one reported in Mou et al., 2012,but the protocol has enhanced efficiency of NKX2.1+ cell production.

In addition, the inventors have demonstrated that this approach iseffective in multiple human iPS cells and ESC cells. Step IV allows thematuration of immature lung progenitors into the NKX2.1+SOX2+p63+CK5+airway stem cells and can also be used to expand such populations toincreased passages and/or to increase the cell quantity for e.g.,functional assays, disease modeling and drug screening.

Example 4: The Combination of BMP7 and FGF7 Signaling, as Well as WNTand MAPKK/ERK Inhibition, Generates Proximal Airway Progenitors

As demonstrated herein above, most ESC-derived Nkx2.1+ cells at day 9were immature lung endoderm cells. Therefore, these cells weredifferentiated in order to produce more mature Nkx2.1+Sox2+ embryonicairway progenitors. The inventors selectively applied signaling factorsto generate airway stalk progenitors from immature lung Nkx2.1+ cells(FIGS. 5A and B). The cells were switched at day 9 to a “proximalinduction” medium prepared with RA-supplemented B27, BMP7, FGF7, IWR-1(WNT antagonist), and Noggin (data not shown). After 2-3 days, anincrease in Nkx2.1+Sox2+ airway progenitor cells was observed. Theproportion was ˜10% of the total number of Nkx2.1+ cells, as compared to1%-2% before treatment. In separate experiments in which individualgrowth factors were removed, it was found that WNT antagonism had themost pronounced effect on Nkx2.1+Sox2+ cell production. Addition ofDorsomorphin (BMP antagonist) to replace Noggin at this stage had littleeffect on the number of Nkx2.1+Sox2+ cells. Interestingly, the smallmolecule PD98059 (a MAPKK/ERK inhibitor) enhanced generation ofNkx2.1+Sox2+ airway progenitor cells to up to 18% of the total number ofNkx2.1+ cells (data not shown). These data indicate that airwayprogenitors are formed after inhibition of a MAPKK/ERK-related pathway.Most notably, the inventors started to detect a small fraction ofNkx2.1+p63+ cells (about 1%-4% of the total number of Nkx2.1+ cells) atday 11-12 (FIG. 6D). An increase in the duration of culture in the“proximal induction” medium from 2 to 5 or more days resulted in higherproportions of Nkx2.1+Sox2+ and Nkx2.1+p63+ cells (data not shown).Although the production of Nkx2.1+Sox2+ and Nkx2.1+p63+ cells stillneeds to be optimized, these results demonstrate that ESC-derivedNkx2.1+ cells can be differentiated into proximal airway progenitors andconducting airway basal cells. An approach for isolating specificsub-populations of these cells, e.g., Nkx2.1+, Sox2+ cells or Nkx2.1+,Sox9+ cells or Nkx2.1+, p63+ cells is to transfect the cells with areporter gene construct, e.g., a GFP reporter construct driven by Sox2,Sox9 or p63 promoter. Expression of GFP under these circumstances willidentify the cell as permissive for expression of the subject factor. Itis contemplated that these studies can be performed on cells at limitingdilution. For example, a population of anterior foregut endoderm cellscan be induced to differentiate to Nkx2.1+, Tuj1−, Pax8−, multipotentlung progenitors as described herein, followed by treatment todifferentiate to airway progenitors as also described. After appropriatetime under inducing conditions, the population can be subjected tolimiting dilution and plated at one cell per well in a microtiter dish.The isolated cells can be permitted to clonally expand in culture, and aportion of expanded cells can be tested, e.g., via immunofluorescence,for the expression or co-expression of the derived airway progenitormarkers, e.g., Nkx2.1, Sox2, Sox9, p63 or even more distaldifferentiation markers, e.g., CCSP, FoxJ1, etc. In this manner isolatedpopulations of the various progenitors can be prepared.

One of skill in the art will recognize that alternate methods ofproducing an isolated population of cells can also be employed with themethods described herein. In some embodiments, a chemical or combinationof chemicals can be used to enhance the efficiency of celldifferentiation in culture to increase the numbers of a desired celltype. Such enhanced efficiency of cell differentiation permits theproduction of an isolated population of the desired human lungprogenitor cell type by e.g., positive selection for a desired cellphenotype or selective killing of a non-desired cell phenotype.Alternatively, the cells can be genetically modified such that theyexpress GFP upon differentiation to a desired human lung cell progenitorphenotype, permitting the use of e.g., FACS sorting to isolate cells ofa defined phenotype. In addition, one of skill in the art can generateantibodies (e.g., monoclonal antibodies) against a particular cellsurface marker or combination of markers. Such an antibody orcombination of antibodies can be used to purify a desired cell type froma population of cells.

Example 5: ESC-Derived Nkx2.1+ Cell Populations can Differentiate intoMature Airway Epithelium when Transplanted In Vivo

Nkx2.1+ lung progenitor cells derived from pluripotent stem cells shouldpossess the capacity to generate functional respiratory epithelium to beuseful. Therefore, mouse ESC-derived Nkx2.1+ progenitors were tested fortheir capacity to form mature respiratory epithelium by subcutaneousengraftment (data not shown). The assay evaluated the ability of Nkx2.1+cells to differentiate within a mixed cell population. 20,000-50,000cells were suspended in 50% Matrigel and injected under the skin ofimmunodeficient mice. 20-30 days after injection, engrafted tissues wereexcised for examination. Differentiation of airway epithelium from mouseESC-derived lung endoderm was observed upon subcutaneous engraftment.Immunofluorescence staining indicates that some Nkx2.1+ cells arepositive for SOX2 (data not shown). Immunofluorescence staining furtherindicated differentiation of ESC-derived Nkx2.1+ cells into p63+ airwaybasal stem cells, CC10+ Clara cells, FoxJ1+ ciliated cells and Muc5ac+goblet cells (data not shown).

It was observed that many epithelial spheres formed within grafts andthat some of these spheres contained Nkx2.1-expressing cells. In someNkx2.1+ spheres, markers of mature airway epithelial cells (data notshown) were detected, including Sox2+ proximal airway epithelial cells(data not shown), p63+ basal stem cells, CC10+ Clara cells, FoxJ1+ciliated cells, and Muc5ac+ mucin-secreting cells (data not shown).

Triple immunofluorescence staining with confocal imaging demonstratedspheres that contain more than one marker of mature airway epithelium.Such mature airway epithelial markers have never been detected inESC-derived teratomas. Previously published basal stem celldifferentiation using a three-dimensional sphere-forming assay producedciliated and basal cells but not Clara cells (Rock et al., 2009). Theinventors did not detect any type I and type II pneumocyte markers suchas PRO-SPC, PRP-SPA and AQUAPORIN5 despite having distal lungmultipotent cells (Nkx2.1+Sox9+ and Nkx2.1+FoxP2+) in the initial cellmixture. Overall, it was concluded that ESC-derived Nkx2.1+cell-containing populations are capable of airway epithelial celldifferentiation.

Example 6: An Efficient and Reproducible Step-Wise Approach to GenerateNKX2.1+ Lung Cells from Human Cystic Fibrosis iPS Cells

Next, it was examined whether a similar step-wise differentiationapproach could be used to generate lung airway progenitors from CysticFibrosis (CF) disease-specific human iPSCs (FIG. 6). All differentiationsteps were performed in xeno-free conditions. Furthermore, two of the CFiPSC lines were generated using modified RNAs (RiPS) rather than virallyencoded reprogramming factors (Warren et al., 2010) and thus weregenetically unmodified, another advantage for possible future clinicaluse. CF iPSCs were maintained on Geltrex-coated plates in completemTeSR1 medium. High yields of definitive endoderm (DE) were obtainedafter treatment for 3-4 days in RPMI-1640 media in the presence ofActivin (100 ng/ml) and PI3 kinase inhibitor LY294002 (5 μM). More than85%-90% of cells co-expressed the transcription factors SOX17 and FOXA2at day 4, demonstrating very efficient production of DE from a RiPS cellline that is compound heterozygous for CFTR mutant alleles Δ508 andG551D (data not shown). This protocol was also successfully applied toother human pluripotent cells including three Cystic Fibrosis iPSC lineshomozygous for the Δ508 allele, a wild-type human BJ RiPS cell line(Warren et al., 2010), and the HUES-3 and HUES-9 human ESC lines (datanot shown).

By using anteriorization conditions similar to Green et al., theinduction of SOX2 expression in DE cells was obtained with efficienciesof up to 50%-60% after 4 days of treatment with A-83-01 (a TGFβantagonist; data not shown). It was found that Noggin (a BMP4antagonist) was actually not necessary for this anteriorization. Similarcombinations of growth factors and agonists (BMP4, FGF2 and GSK3iXV)identified in the studies on mouse ES cells can be used to generateNKX2.1+ cells with efficiencies of 10%-30% (data not shown). TheseNKX2.1+ cells were negative for TUJ1 and PAX8 (data not shown),demonstrating that they were not of neural or thyroid identity. Inaddition, subpopulations of these NKX2.1+ cells were also positive forSOX2 and SOX9 (data not shown). These markers suggest the presence ofboth committed airway progenitors (NKX2.1+SOX2+ cells) and multipotentembryonic lung progenitors (NKX2.1+SOX9+ cells).

The expression of various anterior cell fate genes were quantified byqPCR (FIG. 9) and similar gene expression patterns were observed ascompared to the expression patterns observed during mouse ES celldifferentiation. SOX2 was downregulated after differentiation intodefinitive endoderm and then increased as expected afteranteriorization. A dramatic upregulation of NKX2.1, SOX9, and FOXP2expression (20-30 fold) in anteriorized endoderm cells was observedafter FGF2/BMP4/WNT induction. The expression of other anterior cellfate genes including PAX8 (thyroid endoderm), PAX9 (pharyngealendoderm), and TBX1 (pharyngeal endoderm anterior to lung/esophagus) wasonly modestly upregulated without any expression of FOXN1 (thymusendoderm). In aggregate, the differentiation spectrum of the endodermwas remarkably similar in the mouse and human platforms.

Finally, human RiPS cell-derived NKX2.1+ mixed cell populations weresubcutaneously engrafted (data not shown). Many spheres formed inengrafted tissues after 30 days under the skin of immunodeficientrecipient mice. In NKX2.1+ spheres, some of the NKX2.1+ cellsco-expressed p63, indicating that these NKX2.1+ cells had matured intoairway basal stem cells (data not shown).

Example 7: Summary

Herein is reported a step-wise strategy to differentiate pluripotentstem cells into lung multipotent progenitors (Nkx2.1+Sox9+ andNkx2.1+FoxP2+) and airway progenitors (Nkx2.1+Sox2+). The inventors showthat mimicking the regionalization of the embryonic foregut enhanced thesubsequent differentiation of Nkx2.1+ lung cells. This lends credence tothe notion that the optimization of each discrete step duringembryogenesis will result in an improvement in the efficiency ofdifferentiation at each subsequent step. Some of these Nkx2.1+ lungendoderm cells were ultimately differentiated into multipotent embryoniclung progenitor cells and airway progenitor cells, a finding that hasnot been previously reported. Additionally, the committed Nkx2.1+ lungprogenitor cells produced specific mature cell markers of the airwayepithelium when engrafted. The inventors then adapted their strategy toproduce disease-specific lung progenitor cells from human CysticFibrosis iPS cells and other human pluripotent stem cell lines, thuscreating a new platform for dissecting human lung disease.

Additionally, ESC systems can be used as a discovery tool to investigatesteps in lung development. It was also demonstrated that FGF signalingin addition to BMP and WNT signaling is required in the current culturesystem for NKX2.1 induction, an observation that has proven difficult todefine in murine genetic systems due to the presence of multiple FGFs inthe vicinity of the foregut lung anlage. Mechanistic dissection of theBMP4 signaling effect also revealed a novel observation that BMP4signaling occurs through a Smad-dependent pathway and that pharmacologicSmad modulation enhances Nkx2.1+ lung cell differentiation. Theinventors have also shown that the carefully timed staged antagonism oragonism of the same pathway is essential to drive optimaldifferentiation. The early addition of WNT signals that are laternecessary for Nkx2.1+ lung cell induction at the endoderm stage, forexample, regionalizes endoderm to a hindgut intestinal epithelium thatis refractory to forming Nkx2.1+ cells (Spence et al., 2011; Cao et al.,2011; Sherwood et al., 2011). Thus not only combinations of signalingcascades but also their precise timing is essential to recapitulate invivo differentiation. Accordingly the system described herein is auseful adjunctive platform for defining mechanisms of lung celldifferentiation that were not previously described using mousedevelopmental genetics.

This work was the first to demonstrate the Nkx2.1+Sox2+ proximal airwayprogenitors as well as the Nkx2.1+p63+ double-positive airway stem cellsthat are derived from both human and mouse pluripotent stem cells. Zaretand colleagues have suggested that the anterior gut contains anepigenetic pattern that regulates the ability of gut endoderm todifferentiate into a particular tissue despite the presence of thecorrect growth factors (Zaret et al., 2008). It will be of interest toinvestigate whether an endodermal pre-pattern based on epigeneticfactors could be modulated to enhance lung progenitor cellspecification.

The inventors have successfully differentiated the mixed cellpopulations into airway epithelium by engrafting them into animmunodeficient mouse subcutaneously. Indeed, it is possible that othercells within this mixed population are necessary to induce airwayprogenitors to form the respiratory spheres.

In summary, provided herein is a robust and generally applicableprotocol for DE generation, endoderm anteriorization to foregut, andderivation of the earliest lung Nkx2.1+ endoderm as well as multipotentNkx2.1+Sox9+ lung progenitors, Nkx2.1+Sox2+ embryonic airwayprogenitors, and Nkx2.1+p63+ airway stem cells from mouse ESCs. Thestrategy was adapted to generate disease-specific lung progenitor cellsfrom human Cystic Fibrosis iPSCs, thus establishing a new platform fordissecting human lung disease. This allows for disease modeling, drugscreening, genetic rescue of the mutant CFTR gene (using homologousrecombination, zinc finger nucleases, or TAL effector nucleases) andautologous transplantation. Furthermore, this strategy can in principlebe applied to any human genetically influenced lung disorder. Given thewealth of emerging data in the genetics of common human lung diseases,this platform will also serve as a springboard to test the biologicalsignificance of candidate genes in airway epithelium. Finally, theability to generate a large number of lung epithelial cells can enablebiochemical and proteomic experiments not previously possible due to thelimited supply of human lung epithelial cells.

Example 8: Experimental Procedures

Mice and Human Cell Lines:

NOD-SCID IL2Rgamma null (NOD/SCIDIl2rg−/−) immunodeficient mice werepurchased from Jackson Laboratory. Animal procedures were performed inaccordance with Massachusetts General Hospital (MGH) and nationalguidelines and regulations and approved by the Institutional Animal Careand Use Committee (IACUC) for MGH. The use of human Cystic Fibrosisinduced-pluripotent cell lines and embryonic cell lines (Hues-3 andHues-9) were reviewed and approved by the Embryonic Stem Cell ResearchOversight committee (ESCRO) and IRB at MGH.

Mouse Endoderm Anteriorization and Nkx2.1 Cell Differentiation:

Mouse definitive endoderm was generated as described herein. Toanteriorize endoderm, on and after day 5, the cells were split andre-seeded on to the plates pre-coated with 804G-conditioned medium andfed with D0 medium+0.5-1 μM A8301 (CalBiochem, 616454) for 2 days. Thenthe media was rinsed with D0 medium 2 times and switched to D0 mediumsupplemented with 50 ng/ml BMP4, 100 ng/ml FGF2 (GIBCO, PHG0026), and5-10 nM GSK3iXV for another 2-3 days. To generate Nkx2.1+Sox2+ proximalprogenitor cells, the cells were rinsed with D0 medium 2 times andswitched to D0 medium containing RA-supplemented B27, 50 ng/ml BMP7, 50ng/ml FGF7, 100 nM IWR-1, and 1-2 μM PD98059 for 2 days or longer times.

Human iPSC Culture and Differentiation:

Human iPSCs were maintained on Geltrex-coated plates in complete mTeSR1medium (Stemcell). High yields of definitive endoderm progenitors wereobtained after treatment for 3-4 days in RPMI-1640 media in the presenceof 2% B27 supplement minus vitamin A (GIBCO, 12587-010), Activin A(Peprotech, 100 ng/ml) and PI3 Kinase inhibitor LY294002 (5 μM). Togenerate foregut endoderm cells, definitive endoderm was treated for 4days with RPMI-1640 medium containing 2% B27, 500 nM A-83-01 (TGFβantagonist) with or without Noggin (BMP4 antagonist, 100 ng/ml) orDorsomorphin (2-5 μM). After that, the cells were exposed for 4 days orlonger time to RPMI-1640 medium containing 2% B27, 50 ng/ml BMP4, 100ng/ml FGF2 and 5 nM GSK3iXV for Nkx2.1 induction from endoderm cells.

Immunofluorescence:

At each differentiation step, the cells were fixed with freshparaformaldehyde (4%) for 15 min at room temperature, rinsed in PBS,washed with PBS+0.2% Triton X-100 and incubated with the primaryantibodies at 4° C. overnight (>16 hrs) diluted in PBS+1% BSA. Followingincubation, the cells were rinsed 4 times PBS+0.2% Triton X-100, andincubated with secondary antibodies at room temperature for 2 hours. Theimages were visualized using an Olympus IX71 inverted fluorescencemicroscope or a Nikon A1 Confocal Laser microscope. The primaryantibodies used are summarized in the following table:

TABLE 1 Primary Antibody List Antibody Source SOX2 R&D, goat polyclonal,AF2018 SOX9 R&D, goat polyclonal, AF3075 SOX17 R&D, goat polyclonal,AF1924 FOXP2 R&D, goat polyclonal, AF5647 FOXA2 Santa Cruz technology,goat polyclonal, sc-655 TUJ1 Sigma, mouse monoclonal, T8578 PAX6Developmental Studies Hybridoma Bank, mouse monoclonal PAX8 Abcam, mousemonoclonal, ab53490 NKX2.1 Abcam, rabbit polyclonal, ab76013 P63 SantaCruz Technology, mouse monoclonal, sc-56188 CDX2 BioGenex, mousemonoclonal, CDX2-88 KI67 BD pharmaceutical, mouse monoclonal, 556003CCSP From Dr. Barry Stripp, Duke University Medical Center, goatpolyclonal FOXJ1 eBioscience, mouse monoclonal, 14-9965-82 MUC5AC ThermoScientific, mouse monoclonal, 2013-05

The secondary antibodies were purchased from Invitrogen (AlexaFluor-549and AlexaFluor-488). The quantification was performed by counting atleast 5 random fields at 20× magnification and calculating the averageand standard deviation.

Embryo Harvest, Section Cutting and Staining:

The embryos or lungs at desired embryonic stages were dissected out andfixed at 4° C. for 6 hours or overnight with fresh 4% PFA. Then tissueswere rinsed with PBS for 3 min (5 min per time) and incubated in 30%sucrose in PBS at 4° C. overnight. The tissues were soaked in OCT for 1hour and then frozen in OCT for cryosectioning at 7 μm thickness. Theslides were stained with the primary and secondary antibodies asdescribed above.

Differentiation of Mouse ESC and Human iPS-Derived Nkx2.1+ LungProgenitors after Subcutaneous Engraftment:

2-5×10⁵ cells were suspended in 200 μl 1:1 mixture of Growth FactorReduced Matrigel (BD Biosciences, 354230) Advanced DMEM and injectedsubcutaneously into NOD-SCID IL2Rgamma null mice. The tissues wereharvested and examined after 20-30 days, fixed overnight in cold 4%paraformaldehyde, rinsed 2 times with PBS, soaked in 30% sucrose for 2-3hours and then embedded in OCT, and sectioned at 7 μm. The slides wereexamined for airway epithelial cells based on NKX2.1 expression. Thedifferentiation of ESC-derived Nkx2.1+ cells into basal stem cells wasbased on expression of P63 and CK5, goblets cells based on expression ofMUC5AC, Clara cells based on expression of CCSP, and ciliated cellsbased on the expression of FOXJ1.

Exemplary Media for Differentiation

Step I: From Human ES Cells and iPS Cells to Definitive Endoderm

An exemplary RPMI-1640 based differentiation medium can contain 2% B-27(retinoic acid free), 0.1% Albumax II, 1× Glutamax, 1× non-essentialamino acids (NEAA), 5 uM LY294002 (PI3K inhibitor), and 100 ng/mlActivin A. In some embodiments, 5 μM LY294002 and/or 100 ng/mL Activin Aare used. However, LY294002 can be used within a range of e.g., 2-10 uMand Activin A can be used within a range of e.g., 75-150 ng/ml. In someembodiments, the length of time for treatment for Step I is 4-5 days.

Step II: From Definitive Endoderm to Anterior Foregut Endoderm

An RPMI-1640 based differentiation medium for Step II can contain 2%B-27 (retinoic acid free), 0.1% Albumax II, 1× Glutamax, 1×non-essential amino acids (NEAA), 0.5-2 uM TGFb antagonist A8301,100-500 nM WNT antagonist IWR-1. In some embodiments, the length of timefor treatment is 2-4 days.

Step III: From Anterior Foregut Cells to Nkx2.1+ Cells

An exemplary RPMI-1640 based differentiation medium for Step III cancontain 2% B-27 (supplement with RA), 0.1% Albumax II, 1× Glutamax, 1×non-essential amino acids (NEAA), 20-200 nM BMP4, 20-200 ng/ml FGF2,5-50 nM GSK3iXV (or 100-1000 nM CHIR-99021 to replace GSK3iXV). In someembodiments, the length of time for treatment is about 4-6 days.

Step IV: from Nkx2.1 Early Lung Endoderm Cells to Nkx2.1+Sox2+ ProximalAirway and Nkx2.1+p63+ Airway Basal Stem Cells

An exemplary RPMI-1640 based medium can contain 2% B-27 (supplement withRA), 0.1% Albumax II, 1× Glutamax, 1× non-essential amino acids (NEAA),20-100 ng/ml BMP7, 20-100 ng/ml FGF7, 50-100 ng/ml IWR-1 (WNTantagonist) and 1-2 uM PD98059 (MAPKK/ERK antagonist). In someembodiments, the length of time for treatment is about 4-6 days.

A second exemplary medium for differentiation is depicted herein in FIG.12.

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The invention claimed is:
 1. A method of generating a human lungprogenitor cell or population of human lung progenitor cells that isNkx2.1 positive, Tuj1 negative and Pax8 negative, the method comprisingcontacting a human foregut endoderm cell with FGF2, WNT and BMP4, eachfor a time sufficient and at a concentration sufficient to permitdifferentiation of said human foregut endoderm cell to an Nkx2.1positive, Tuj1 negative, Pax8 negative lung progenitor cell.
 2. Themethod of claim 1, wherein the contacting is performed for at least 2days.
 3. The method of claim 1, further comprising contacting the Nkx2.1positive, Tuj1 negative, Pax8 negative lung progenitor cell with BMP7,FGF7, a WNT antagonist, and a MAPKK/ERK antagonist, each for a timesufficient and at a concentration sufficient to permit differentiationof the Nkx2.1 positive, Tuj1 negative, Pax8 negative lung progenitorcell to a Nkx2.1 positive, Sox2 positive proximal airway multipotentprogenitor cell.